IL-12 heterodimeric Fc-fusion proteins

ABSTRACT

The present invention provides novel IL-12 Fc fusion proteins, methods of making and using the same. The IL-12 Fc fusion proteins are useful for treatment of cancer and can be used in combination with checkpoint blockade.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/718,087, filed Apr. 11, 2022, which is a continuation of U.S. patentapplication Ser. No. 16/592,656, filed Oct. 3, 2019, now U.S. Pat. No.11,358,999, which claims the benefit of U.S. Provisional Application No.62/740,813, filed Oct. 3, 2018, U.S. Provisional Application No.62/810,038 filed Feb. 25, 2019, and U.S. Provisional Application No.62/848,512 filed May 15, 2019 all of which are incorporated herein byreference in their entirety for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 21, 2019, isnamed 067461-5225-US_SL.txt and is 1,640,919 bytes in size.

BACKGROUND OF THE INVENTION

In order for T cells to mount an effective anti-tumor response, threethings must occur. T cells must first engage antigenic tumor peptidespresented by MHC in the tumor environment. Second, costimulatorymolecules must bind to the T cells. And third, the T cells must beinduced by cytokines such as IL-12 and IL-2 to produce costimulatorycytokines such as IFNγ which allows differentiation and expansion.Recognition of tumor peptides alone in the absence of cytokine inductionleads to T cells becoming anergic, thereby leading to tolerance.Accordingly, a very promising approach in cancer immunotherapy iscytokine-based treatments. In fact, IL-2 has been approved for use inpatients with metastatic renal-cell carcinoma and malignant melanoma.However, there are currently no approved uses of recombinant IL-12 inhumans.

Recombinant IL-12 is a promising cytokine-based treatment due to itsbroad effect in activating the immune system. However, IL-12 has thusfar faced hurdles in human clinical trials due to toxicity. As withother cytokines, the short half-life of IL-12 requires frequent bolusinjections.

Additionally, IL-12 is composed of an α-chain (the p35 subunit;IL-12p35) and a β-chain (the p40 subunit; IL-12p40) covalently linked toform the biologically active IL-12 heterodimer. IL-12 exerts its cellsignaling function through binding by binding to a dimeric IL-12receptor complex composed of IL-12 receptor β1 (IL-12Rβ1) and IL-12receptor β2 (IL-12Rβ2) on T cells and inducing IFNγ secretion. However,the IL-12p40 subunit can also exist as a homodimer which has beenreported to antagonize IL-12 activity by competing for binding to IL-12receptor.

The present invention addresses the short half-life of IL-12 byproviding novel IL-12-Fc fusion proteins, as well as novel IL-12variants with decreased potency.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides a heterodimeric Fc fusionprotein comprising: a) a first fusion protein comprising a variantIL-12p40 subunit domain and a first Fc domain, wherein said IL-12p40subunit domain is covalently attached to the N-terminus of said first Fcdomain; and b) a second fusion protein comprising an IL-12p35 subunitdomain and a second Fc domain, wherein said IL-12p35 subunit domain iscovalently attached to the N-terminus of said Fc domain; wherein saidfirst and said second Fc domains comprise modifications promotingheterodimerization of said first and said second Fc domains. In someembodiments, the present invention provides a heterodimeric Fc fusionprotein, wherein said IL-12p40 subunit has one or more amino acidsubstitutions selected from the group consisting of: E59K, E59Q, D18N,D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, D62N, E73Q,D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N,R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N,K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein, wherein said IL-12p40 subunit has amino acidsubstitutions selected from the group consisting ofN103D/N113D/N200D/N281D, Q42E/E45Q, E45Q/Q56E, Q42E/E59Q, Q56E/E59Q,Q42E/E45Q/Q56E, E45Q/Q56E/E59Q, E32Q/E59Q, D34N/E59K, D34N/E59K/K99E,D34K/E59K/K99E, E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q,E59Q/E187Q, S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E,E59Q/K260E, E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y,E59Y/K99Y, E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein, wherein said IL-12p40 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:57(IL-12p40(N103D)), ii) SEQ ID NO:58 (IL-12p40(N113D)), iii) SEQ ID NO:59(IL-12p40(N200D)), iv) SEQ ID NO:60 (IL-12p40(N281D)), v) SEQ ID NO:61(IL-12p40(N103D/N113D/N200D/N281D)), vi) SEQ ID NO:62 (IL-12p40(Q42E)),vii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)),ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi)SEQ ID NO:67 (IL-12p40(Q42E/E45Q)), xii) SEQ ID NO:68(IL-12p40(E45Q/Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E/E59Q)), xiv)SEQ ID NO:70 (IL-12p40(Q56E/E59Q)), xv) SEQ ID NO:71(IL-12p40(Q42E/E45Q/Q56E)), xvi) SEQ ID NO:72(IL-12p40(E45Q/Q56E/E59Q)), xvii) SEQ ID NO:73 (IL-12p40(D161N)), xviii)SEQ ID NO:74 (IL-12p40(E73Q)), xix) SEQ ID NO:75 (IL-12p40(Q144E)), xx)SEQ ID NO:76 (IL-12p40(E262Q)), xxi) SEQ ID NO:77 (IL-12p40(E100Q)),xxii) SEQ ID NO:78 (IL-12p40(D18N)), xxiii) SEQ ID NO:79(IL-12p40(E33Q)), xxiv) SEQ ID NO:80 (IL-12p40(Q229E)), xxv) SEQ IDNO:81 (IL-12p40(E235Q)), xxvi) SEQ ID NO:82 (IL-12p40(Q256N)), xxvii)SEQ ID NO:83 (IL-12p40(E299Q)), xxviii) SEQ ID NO:84 (IL-12p40(D87N)),xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii)IL-12p40(S43K), xxxiii) SEQ ID NO:379 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E)), xxxiv) SEQ ID NO:205(IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii)IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl)SEQ ID NO:206 (IL-12p40(E32Q/E59Q)), xli) SEQ ID NO:207(IL-12p40(D34N/E59Q)), xlii) SEQ ID NO:208 (IL-12p40(E59Q/E187Q)),xliii) SEQ ID NO:209 (IL-12p40(S43E/E59Q)), xliv) SEQ ID NO:210(IL-12p40(S43K/E49Q)), xlv) SEQ ID NO:211 (IL-12p40(E59Q/K163E)), xlvi)SEQ ID NO:212 (IL-12p40(E59Q/K99E)), xlvii) SEQ ID NO:213(IL-12p40(E59Q/K258E)), xlviii) SEQ ID NO:214 (IL-12p40(E59Q/K260E)),xlix) SEQ ID NO: 326 (IL-12p40 (D34N/E59K)), 1) SEQ ID NO: 325(IL-12p40(E59K/K99E)), li) SEQ ID NO: 339 (IL-12p40(D18K/E59K/K99E)),lii) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), liii) SEQ ID NO: 336(IL-12p40 (E59K/K99Y)), liv) SEQ ID NO: 335 (IL-12p40 (E59Y/K99E)), lv)SEQ ID NO: 338 (IL-12p40 (E45K/E59K/K99E)), lvi) SEQ ID NO: 340(IL-12p40 (E59K/K99E/Q144E)), lvii) SEQ ID NO: 341 (IL-12p40(E59K/K99E/Q144K)), lviii) SEQ ID NO: 342 (IL-12p40 (E59K/K99E/R159E)),lix) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), lx) SEQ ID NO: 344(IL-12p40 (D18K/E59K/K99E/K264E)), lxi) SEQ ID NO: 360 (IL-12p40(C252S)), lxii) SEQ ID NO: 361 (IL-12p40 (DI8K/E59K/K99E/C252S)), lxiii)SEQ ID NO: 362 (IL-12p40 (D18K/E59K/K99E/C252S/K264E)), lxiv) SEQ ID NO:363 (IL-12p40 (E59K/K99Y/C252S)), lxv) SEQ ID NO: 364 (IL-12p40(E59K/K99E/C252S/K264E)), lxvi) SEQ ID NO: 365 (IL-12p40(E59K/K99E/C252S)), lxvii) SEQ ID NO: 254 (IL-12p40 (N103D/N113D)),lxviii) SEQ ID NO: 255 (IL-12p40 (N103D/N200D)), lxix) SEQ ID NO: 256(IL-12p40 (N103D/N281D)), lxx) SEQ ID NO: 257 (IL-12p40 (N113D/N200D)),lxxi) SEQ ID NO: 258 (IL-12p40 (N113D/N281D)), lxxii) SEQ ID NO: 259(IL-12p40 (N200D/N281D)), lxxiii) SEQ ID NO: 260 (IL-12p40(N103D/N113D/N200D)), lxxiv) SEQ ID NO: 261 (IL-12p40(N103D/N113D/N281D)), lxxv) SEQ ID NO: 262 (IL-12p40(N103D/N200D/N281D)), lxxvi) SEQ ID NO: 263 (IL-12p40(N113D/N200D/N281D)), lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)),lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)), lxxix) SEQ ID NO: 266(IL-12p40 (N200Q)), lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)), lxxxi) SEQID NO: 268 (IL-12p40 (N103Q/N113Q)), lxxxii) SEQ ID NO: 269 (IL-12p40(N103Q/N200Q)), lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q/N281Q)),lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q/N200Q)), lxxxv) SEQ ID NO: 272(IL-12p40 (N113Q/N281Q)), lxxxvi) SEQ ID NO: 273 (IL-12p40(N200Q/N281Q)), lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q/N113Q/N200Q)),lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q/N113Q/N281Q)), lxxxix) SEQ IDNO: 276 (IL-12p40 (N103Q/N200Q/N281Q)), xc) SEQ ID NO: 277 (IL-12p40(N113Q/N200Q/N281Q)), xci) SEQ ID NO:278 (IL-12p40(N103Q/N113Q/N200Q/N281Q)), xcii) SEQ ID NO:327 (IL-12p40(D34N/E59K/K99E)), xciii) SEQ ID NO:328 (IL-12p40 (D34K/E59K/K99E)),xciv) SEQ ID NO:329 (IL-12p40 (E32Q/D34N/E59K/K99E)), xcv) SEQ ID NO:331(IL-12p40 (E32K/D34N/E59K/K99E)), xcvi) SEQ ID NO: 337 (IL-12p40(E59Y/K99Y)), xcvii) SEQ ID NO:366 (IL-12p40(E59K/K99E/N103Q/C252S/K264E)), xcviii) SEQ ID NO:367 (IL-12p40(E59K/K99E/N113Q/C252S/K264E)), xcix) SEQ ID NO:368 (IL-12p40(E59K/K99E/N200Q/C252S/K264E)), c) SEQ ID NO:369 (IL-12p40(E59K/K99E/N281Q/C252S/K264E)), ci) SEQ ID NO:370 (IL-12p40(E59K/K99E/N103Q/N113Q/C252S/K264E)), cii) SEQ ID NO:371 (IL-12p40(E59K/K99E/N103Q/N200Q/C252S/K264E)), ciii) SEQ ID NO:372 (IL-12p40(E59K/K99E/N103Q/N281Q/C252S/K264E)), civ) SEQ ID NO:373 (IL-12p40(E59K/K99E/N113Q/N200Q/C252S/K264E)), cv) SEQ ID NO:374 (IL-12p40(E59K/K99E/N113Q/N281Q/C252S/K264E)), cvi) SEQ ID NO:375 (IL-12p40(E59K/K99E/N200Q/N281Q/C252S/K264E)), cvii) SEQ ID NO:376 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E)), cviii) SEQ ID NO:377(IL-12p40 (E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E)), and cix) SEQ IDNO:378 (IL-12p40 (E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E)).

In some embodiments, the present invention provides a heterodimeric Fcfusion protein, wherein said variant IL-12p40 subunit has at least 90%identity to a polypeptide sequence selected from the group consisting ofSEQ ID NO:3 (human IL-12 subunit beta (IL-12p40) precursor sequence) andSEQ ID NO:4 (human IL-12 subunit beta (IL-12p40) mature form sequence),and/or said IL-12p35 subunit has a polypeptide sequence selected fromthe group consisting of SEQ ID NO:1 (human IL-12 subunit alpha(IL-12p35) precursor sequence) and SEQ ID NO:2 (human IL-12 subunitalpha (IL-12p35) mature form sequence).

In some embodiments, the present invention provides a heterodimeric Fcfusion protein, wherein said IL-12p35 subunit is a variant IL-12p35subunit.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein, wherein said IL-12p40 subunit is a variant IL-12p40subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex; and/or said IL-12p35 subunit is a variant IL-12p35subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein, wherein said variant IL-12p35 subunit has one or moreamino acid substitutions selected from the group consisting of N21D,Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A,F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D,N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, andN195Q.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein, wherein said variant IL-12p35 subunit has amino acidsubstitutions selected from the group consisting of: N71D/N85D/N195D,N151D/E153Q, N151D/D165N, Q130E/N151D, N151D/K158E, E79Q/N151D,D55Q/N151D, N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q, N71Q/N195Q,N85Q/N195Q, N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, and N85D/N195D.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein, wherein said variant IL-12p35 subunit has a polypeptidesequence selected from the group consisting of: i) SEQ ID NO:113(IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115(IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D/N85D/N195D)), v) SEQID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii)SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(Q135E)),ix) SEQ ID NO:121 (IL-12p35(Q35D)), x) SEQ ID NO:122 (IL-12p35(Q146E)),xi) SEQ ID NO:123 (IL-12p35(N76D)), xii) SEQ ID NO:124(IL-12p35(E162Q)), xiii) SEQ ID NO:125 (IL-12p35(E163Q)), xiv)IL-12p35(N21D), xv) SEQ ID NO:333 (IL-12p35(D55Q)), xvi) IL-12p35(E79Q),xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx)SEQ ID NO:227 (IL-12p35(N151K)), xxi) SEQ ID NO:226 (IL-12p35(E153K)),xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO:225(IL-12p35(N151D/E153Q)), xxv) SEQ ID NO:228 (IL-12p35(N151D/D165N)),xxvi) SEQ ID NO:229 (IL-12p35(Q130E/N151D)), xxvii) SEQ ID NO:230(IL-12p35(N151D/K158E)), xxviii) SEQ ID NO:231 (IL-12p35(E79Q/N151D)),xxix) SEQ ID NO:232 (IL-12p35(D55Q/N151D)), xxx) SEQ ID NO:233(IL-12p35(N136D/N151D)), xxxi) SEQ ID NO:234 (IL-12p35(N21D/N151D)),xxxii) SEQ ID NO:235 (IL-12p35(E143Q/N151D)), xxxiii) SEQ ID NO: 345(IL-12p35(F96A)), xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)), xxxv) SEQ IDNO: 347 (IL-12p35(L89A)), xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)),xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)), xxxviii) SEQ ID NO: 350(IL-12p35(L75A)), xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)), xxxv) SEQ IDNO: 279 (IL-12p35 (N71Q)), xxxvi) SEQ ID NO: 280 (IL-12p35 (N85Q)),xxxvii) SEQ ID NO: 281 (IL-12p35 (N195Q)), xxxviii) SEQ ID NO: 282(IL-12p35 (N71Q/N85Q)), xxxix) SEQ ID NO: 283 (IL-12p35 (N71Q/N195Q)),xl) SEQ ID NO: 284 (IL-12p35 (N85Q/N195Q), xli) SEQ ID NO: 285 (IL-12p35(N71Q/N85Q/N195Q)), xlii) SEQ ID NO: 286 (IL-12p35 (N71D/N85D)), xliii)SEQ ID NO: 287 (IL-12p35 (N71D/N195D), xliv) SEQ ID NO: 288 (IL-12p35(N85D/N195D)), xlv) SEQ ID NO: 333 (IL-12p35 (D55Q)), and xlvi) SEQ IDNO: 334 (IL-12p35 (D55K)).

In some embodiments, the present invention provides a heterodimeric Fcfusion protein, wherein said modifications promoting heterodimerizationof said first and said second Fc domains are a set of amino acidsubstitutions selected from the group consisting of L368D/K370S andS364K; L368D/K370S and S364K/E357L; L368D/K370S and S364K/E357Q;T411E/K360E/Q362E and D401K; L368E/K370S and S364K; K370S andS364K/E357Q; T366S/L368A/Y407V and T366W; T366S/L368A/Y407V/Y349C andT366W/S354C, according to EU numbering.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein, wherein said variant IL-12p40 subunit domain is attachedto said first Fc domain using a first domain linker and/or said IL-12p35subunit domain is attached to said second Fc domain using a seconddomain linker.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein, wherein said first and/or said second Fc domains have anadditional set of amino acid substitutions comprisingQ295E/N384D/Q418E/N421D, according to EU numbering.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein, wherein said first and/or said second Fc domains have anadditional set of amino acid substitutions selected from the groupconsisting of G236R/L328R, E233P/L234V/L235A/G236_/S239K,E233P/L234V/L235A/G236_/S239K/A327G,E233P/L234V/L235A/G236_/S267K/A327G, E233P/L234V/L235A/G236_, andE233P/L234V/L235A/G236_/S267K, according to EU numbering.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein, wherein said first and second Fc domains furthercomprise amino acid substitutions M428L/N434S.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein, wherein said heterodimeric Fc fusion protein comprises:

-   -   a) said first fusion protein having a polypeptide sequence        selected from the group consisting of: i) SEQ ID NO:47        (XENP27201 Chain 1), ii) SEQ ID NO:85 (XenD24752), iii) SEQ ID        NO:86 (XenD24753), iv) SEQ ID NO:87 (XenD24754), v) SEQ ID NO:88        (XenD24755), vi) SEQ ID NO:89 (XenD24756), vii) SEQ ID NO:90        (XenD24757), viii) SEQ ID NO:91 (XenD24758), ix) SEQ ID NO:92        (XenD24759), x) SEQ ID NO:93 (XenD24760), xi) SEQ ID NO:94        (XenD24761), xii) SEQ ID NO:95 (XenD24762), xiii) SEQ ID NO:96        (XenD24763), xiv) SEQ ID NO:97 (XenD24764), xv) SEQ ID NO:98        (XenD24765), xvi) SEQ ID NO:99 (XenD24766), xvii) SEQ ID NO:100        (XenD24767), xviii) SEQ ID NO:101 (XenD24768), xix) SEQ ID        NO:102 (XenD24769), xx) SEQ ID NO:103 (XenD24770), xxi) SEQ ID        NO:104 (XenD24771), xxii) SEQ ID NO:105 (XenD24772), xxiii) SEQ        ID NO:106 (XenD24773), xxiv) SEQ ID NO:107 (XenD24774), xxv) SEQ        ID NO:108 (XenD24775), xxvi) SEQ ID NO:109 (XenD24776), xxvii)        SEQ ID NO:110 (XenD24777), xxviii) SEQ ID NO:111        (XenD24778), xxix) SEQ ID NO:112 (XenD24792), xxx) SEQ ID NO:215        (XenD25922), xxxi) SEQ ID NO:216 (XenD25923), xxxii) SEQ ID        NO:217 (XenD25924), xxxiii) SEQ ID NO:218 (XenD25925), xxxiv)        SEQ ID NO:219 (XenD25926), xxxv) SEQ ID        NO:220(XenD25927), xxxvi) SEQ ID NO:221 (XenD25928), xxxvii) SEQ        ID NO:222 (XenD25929), xxxviii) SEQ ID NO:223        (XenD25930), xxxix) SEQ ID NO:224 (XenD25931), xl) SEQ ID NO:291        (XenD26411), xli) SEQ ID NO:292 (XenD26412), xlii) SEQ ID NO:293        (XenD26413), xliii) SEQ ID NO:294 (XenD26414), xliv) SEQ ID        NO:295 (XenD26415), xlv) SEQ ID NO:296 (XenD26416), xlvi) SEQ ID        NO:297 (XenD26417), xlvii) SEQ ID NO:298 (XenD26418), xlviii)        SEQ ID NO:301 (XenD27070), xlix) SEQ ID NO:302 (XenD27071), 1)        SEQ ID NO:303 (XenD27072), li) SEQ ID NO:304 (XenD27073), lii)        SEQ ID NO:305 (XenD27074), liii) SEQ ID NO:306 (XenD27075), liv)        SEQ ID NO:307 (XenD27076), lv) SEQ ID NO:308 (XenD27077), lvi)        SEQ ID NO:309 (XenD27078), lvii) SEQ ID NO:317        (XenD28173), lviii) SEQ ID NO:318 (XenD24876), lix) SEQ ID        NO:320 (XenD27162), lx) SEQ ID NO:321 (XenD27163), lxi) SEQ ID        NO:323 (XenD27164), lxii) SEQ ID NO:324 (XenD27165) lxiii) SEQ        ID NO:357 (XENP31582 Chain 1), lxiv) SEQ ID NO:358 (XENP31583        Chain 1), lxv) SEQ ID NO:359 (XENP31584 Chain 1), lxvi) SEQ ID        NO:380 (XENP32187 Chain 1), lxvii) SEQ ID NO:381 (XENP32188        Chain 1), lxviii) SEQ ID NO:382 (XENP32189, Chain 1), lxix) SEQ        ID NO:425 (XENP32190 Chain 1), lxx) SEQ ID NO:384 (XENP32191        Chain 1), lxxi) SEQ ID NO:385 (XENP32991 Chain 1), lxxii) SEQ ID        NO:386 (XENP32992 Chain 1), lxxiii) SEQ ID NO:387 (XENP32993        Chain 1), lxxiv) SEQ ID NO:388 (XENP32994 Chain 1), lxxv) SEQ ID        NO:389 (XENP32995 Chain 1), lxxvi) SEQ ID NO:390 (XENP32996        Chain 1), lxxvi) SEQ ID NO:391 (XENP32997 Chain 1), lxxvii) SEQ        ID NO:392 (XENP32998 Chain 1), lxxvii) SEQ ID NO:393 (XENP32999        Chain 1), lxxviii) SEQ ID NO:394 (XENP33000 Chain 1), lxxix) SEQ        ID NO:395 (XENP33001 Chain 1), lxxx) SEQ ID NO:396 (XENP33002        Chain 1), lxxxi) SEQ ID NO:397 (XENP33003 Chain 1), lxxxii) SEQ        ID NO:398 (XENP33004 Chain 1), lxxxiii) SEQ ID NO:426 (XENP33005        Chain 1), lxxxiv) SEQ ID NO:427 (XENP33006 Chain 1), lxxxv) SEQ        ID NO:428 (XENP33007 Chain 1), lxxxvi) SEQ ID NO:429 (XENP33008        Chain 1), lxxxvii) SEQ ID NO:429 (XENP33008 Chain 1), lxxxviii)        SEQ ID NO:430 (XENP33009 Chain 1), lxxxix) SEQ ID NO:431        (XENP33010 Chain 1), and xc) SEQ ID NO:383 (XENP33011 Chain 1);        and    -   b) said second fusion protein having a polypeptide sequence        selected from the group consisting of: i) SEQ ID NO:48        (XENP27201 Chain 2), ii) SEQ ID NO:126 (XenD24779), iii) SEQ ID        NO:127 (XenD24780), iv) SEQ ID NO:128 (XenD24781), v) SEQ ID        NO:129 (XenD24782), vi) SEQ ID NO:130 (XenD24783), vii) SEQ ID        NO:131 (XenD24784), viii) SEQ ID NO:132 (XenD24785), ix) SEQ ID        NO:133 (XenD24786), x) SEQ ID NO:134 (XenD24787), xi) SEQ ID        NO:135 (XenD24788), xii) SEQ ID NO:136 (XenD24789), xiii) SEQ ID        NO:137 (XenD24790), xiv) SEQ ID NO:138 (XenD24791), xv) SEQ ID        NO:236 (XenD25911), xvi) SEQ ID NO:237 (XenD25912), xvii) SEQ ID        NO:238 (XenD25913), xviii) SEQ ID NO:239 (XenD25914), xix) SEQ        ID NO:240 (XenD25915), xx) SEQ ID NO:241 (XenD25916), xxi) SEQ        ID NO:242 (XenD25917), xxii) SEQ ID NO:243 (XenD25918), xxiii)        SEQ ID NO:244 (XenD25919), xxiv) SEQ ID NO:245 (XenD25920), xxv)        SEQ ID NO:246 (XenD25921), xxvi) SEQ ID NO:299        (XenD26427), xxvii) SEQ ID NO:300 (XenD26428), xxviii) SEQ ID        NO:311 (XenD27089), xxix) SEQ ID NO:312 (XenD27090), xxx) SEQ ID        NO:313 (XenD27091), xxxi) SEQ ID NO:314 (XenD27092), xxxii) SEQ        ID NO:315 (XenD27093), xxxiii) SEQ ID NO:316 (XenD27094), xxxix)        SEQ ID NO:319 (XenD24877), xl) SEQ ID NO:322 (XenD27166), xli)        SEQ ID NO:421 (XENP31582 Chain 2), xlii) SEQ ID NO:422        (XENP31583 Chain 2), xliii) SEQ ID NO:423 (XENP31584 Chain        2), xliv) SEQ ID NO:402 (XENP32187 Chain 2), xlv) SEQ ID NO:403        (XENP32188 Chain 2), xlvi) SEQ ID NO:404 (XENP32189, Chain        2), xlvii) SEQ ID NO:405 (XENP32190 Chain 2), xlviii) SEQ ID        NO:406 (XENP32191 Chain 2), xlix) SEQ ID NO:407 (XENP32991 Chain        2), l) SEQ ID NO:408 (XENP32992 Chain 2), li) SEQ ID NO:409        (XENP32993 Chain 2), lii) SEQ ID NO:410 (XENP32994 Chain        2), liii) SEQ ID NO:411 (XENP32995 Chain 2), liv) SEQ ID NO:412        (XENP32996 Chain 2), lv) SEQ ID NO:413 (XENP32997 Chain 2), lvi)        SEQ ID NO:414 (XENP32998 Chain 2), lvii) SEQ ID NO:415        (XENP32999 Chain 2), lviii) SEQ ID NO:416 (XENP33000 Chain        2), lix) SEQ ID NO:417 (XENP33001 Chain 2), lx) SEQ ID NO:418        (XENP33002 Chain 2), lxi) SEQ ID NO:419 (XENP33003 Chain        2), lxii) SEQ ID NO:420 (XENP33004 Chain 2), lxiii) SEQ ID        NO:399 (XENP33005 Chain 2), lxiv) SEQ ID NO:400 (XENP33006 Chain        2), lxv) SEQ ID NO:401 (XENP33007 Chain 2), lxvi) SEQ ID NO:289        (XENP33008 Chain 2), lxvii) SEQ ID NO:289 (XENP33008 Chain        2), lxviii) SEQ ID NO:290 (XENP33009 Chain 2), lxix) SEQ ID        NO:352 (XENP33010 Chain 2), and lxx) SEQ ID NO:354 (XENP33011        Chain 2).

In some embodiments, the present invention provides a heterodimeric Fcfusion protein, wherein said heterodimeric Fc fusion protein is selectedfrom the group consisting of XENP27201, XENP28820, XENP28821, XENP28822,XENP28823, XENP28824, XENP28825, XENP28826, XENP28827, XENP28828,XENP28829, XENP28830, XENP28831, XENP28832, XENP28833, XENP28834,XENP28835, XENP28836, XENP28837, XENP28838, XENP28839, XENP28840,XENP28841, XENP28842, XENP28843, XENP28844, XENP28845, XENP28846,XENP28847, XENP28848, XENP28849, XENP28850, XENP28851, XENP28852,XENP29949, XENP29950, XENP29951, XENP29952, XENP30597, XENP30598,XENP30599, XENP30600, XENP30601, XENP30602, XENP30603, XENP30604,XENP30605, XENP30606. XENP30307, XENP30308, XENP30609, XENP31250,XENP31251, XENP31252, XENP31253, XENP31254, XENP31255, XENP31256,XENP31257, XENP31258, XENP31259, XENP31260, XENP31261, XENP31262,XENP31263, XENP31264, XENP31265, XENP31286, XENP31142, XENP31143,XENP31144, XENP31145, XENP31146, XENP31582, XENP31583, XENP31584,XENP32186, XENP32187, XENP32188, XENP32189, XENP32190, XENP32191,XENP32991, XENP32992, XENP32993, XENP32994, XENP32995, XENP32996,XENP32997, XENP32998, XENP32999, XENP33000, XENP33001, XENP33002,XENP33003, XENP33004, XENP33005, XENP33006, XENP33007, XENP33008,XENP33008, XENP33009, XENP33010, and XENP33011.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein, wherein said heterodimeric Fc fusion protein is selectedfrom the group consisting of: XENP31251, XENP31254, XENP31258,XENP32186, XENP32187, XENP32188, XENP32189, XENP32190, and XENP32191.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein, further comprising one or more modifications to theIL-12p40 subunit selected from the group consisting of: N103Q, N113Q,N200Q, and N281Q.

In some embodiments, the present invention provides a compositioncomprising a heterodimeric Fc fusion protein for use in treating cancerin a subject.

In some embodiments, the present invention provides one or more nucleicacids encoding a heterodimeric Fc fusion protein.

In some embodiments, the present invention provides a host cellcomprising said one or more nucleic acids encoding a heterodimeric Fcfusion protein.

In some embodiments, the present invention provides a method of making aheterodimeric Fc fusion protein, said method comprising culturing a hostcell under conditions whereby said heterodimeric Fc fusion protein isproduced.

In some embodiments, the present invention provides a method ofpurifying a heterodimeric Fc fusion protein, said method comprising: a)providing a composition comprising the heterodimeric Fc fusion protein;b) loading said composition onto an ion exchange column; and c)collecting a fraction containing said heterodimeric Fc fusion protein.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidmodifications at amino acid residues selected from the group consistingof E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32,E33, D34, L40, D41, Q42, S43, E45, L47, T54, 155, Q56, K58, E59, F60,G61, D62, Q65, Y66, E73, K84, E86, D87, G88, 189, W90, D93, D97, K99,E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156,R159, D161, N162, K163, D166, D170, Q172, D174, A176, C177, P178, A179,A180, E181, S183, P185, E187, N200, S204, F206, R208, D209, D214, N218,Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F247, S248, C252,Q256, K258, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292,Y293, and E299.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidmodifications as amino acid residues selected from the group consistingof Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63,L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97,L124, M125, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163,D165, I171, R181, 1182, R183, V185, T186, D188, R189, V190, S192, Y193,N195, and A196.

In another aspect, the present invention provides a heterodimeric Fcfusion protein comprising: a) a first fusion protein comprising a firstprotein domain and a first Fc domain, wherein said first protein domainis covalently attached to the C-terminus of said first Fc domain; and b)a second fusion protein comprising a second protein domain and a secondFc domain, wherein said second protein domain is covalently attached tothe C-terminus of said Fc domain; wherein said first and said second Fcdomains comprise modifications promoting heterodimerization of saidfirst and said second Fc domains and wherein said first protein domaincomprises an IL-12p40 subunit and said second protein domain comprisesan IL-12p35 subunit.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said modifications promoting heterodimerizationof said first and said second Fc domains are a set of amino acidsubstitutions selected from the group consisting of L368D/K370S andS364K; L368D/K370S and S364K/E357L; L368D/K370S and S364K/E357Q;T411E/K360E/Q362E and D401K; L368E/K370S and S364K; K370S andS364K/E357Q and T366S/L368A/Y407V:T366W (optionally including a bridgingdisulfide, T366S/L368A/Y407V/Y349C:T366W/S354C), according to EUnumbering.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said first protein domain is attached to saidfirst Fc domain using a first domain linker and/or said second proteindomain is attached to said second Fc domain using a second domainlinker.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said first and/or said second Fc domains have anadditional set of amino acid substitutions comprisingQ295E/N384D/Q418E/N421D, according to EU numbering.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said first and/or said second Fc domains have anadditional set of amino acid substitutions selected from the groupconsisting of G236R/L328R, E233P/L234V/L235A/G236_/S239K,E233P/L234V/L235A/G236_/S239K/A327G,E233P/L234V/L235A/G236_/S267K/A327G, E233P/L234V/L235A/G236_, andE233P/L234V/L235A/G236_/S267K, according to EU numbering.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected form the group consisting of SEQ ID NO:3 (human IL-12 subunitbeta (IL-12p40) precursor sequence) and SEQ ID NO:4 (human IL-12 subunitbeta (IL-12p40) mature form sequence), and/or said IL-12p35 subunit hasa polypeptide sequence selected from the group consisting of SEQ ID NO:1(human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ IDNO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said first and second Fc domains further compriseamino acid substitutions M428L/N434S.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit and/or said IL-12p35 subunit is a variant IL-12p35 subunit.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex; and/or said IL-12p35 subunit is a variant IL-12p35subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidmodifications at amino acid residues selected from the group consistingof E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32,E33, D34, L40, D41, Q42, S43, E45, L47, T54, 155, Q56, K58, E59, F60,G61, D62, Q65, Y66, E73, K84, E86, D87, G88, 189, W90, D93, D97, K99,E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156,R159, D161, N162, K163, D166, D170, Q172, D174, A176, C177, P178, A179,A180, E181, S183, P185, E187, N200, S204, F206, R208, D209, D214, N218,Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F247, S248, C252,Q256, K258, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292,Y293, and E299.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidsubstitutions selected from the group consisting of D18N, D18K, E32Q,E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q,D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N,R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N,K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has amino acidsubstitutions selected from the group consisting ofN103D/N113D/N200D/N281D, Q42E/E45Q, E45Q/Q56E, Q42E/E59Q, Q56E/E59Q,Q42E/E45Q/Q56E, E45Q/Q56E/E59Q, E32Q/E59Q, D34N/E59K, D34N/E59K/K99E,D34K/E59K/K99E, E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q,E59Q/E187Q, S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E,E59Q/K260E, E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y,E59Y/K99Y, E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has amino acidsubstitutions selected from the group consisting ofN103D/N113D/N200D/N281D, Q42E/E45Q, E45Q/Q56E, Q42E/E59Q, Q56E/E59Q,Q42E/E45Q/Q56E, E45Q/Q56E/E59Q, E32Q/E59Q, D34N/E59K, D34N/E59K/K99E,D34K/E59K/K99E, E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q,E59Q/E187Q, S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E,E59Q/K260E, E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y,E59Y/K99Y, E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:57(IL-12p40(N103D)), ii) SEQ ID NO:58 (IL-12p40(N113D)), iii) SEQ ID NO:59(IL-12p40(N200D)), iv) SEQ ID NO:60 (IL-12p40(N281D)), v) SEQ ID NO:61(IL-12p40(N103D/N113D/N200D/N281D)), vi) SEQ ID NO:62 (IL-12p40(Q42E)),vii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)),ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi)SEQ ID NO:67 (IL-12p40(Q42E/E45Q)), xii) SEQ ID NO:68(IL-12p40(E45Q/Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E/E59Q)), xiv)SEQ ID NO:70 (IL-12p40(Q56E/E59Q)), xv) SEQ ID NO:71(IL-12p40(Q42E/E45Q/Q56E)), xvi) SEQ ID NO:72(IL-12p40(E45Q/Q56E/E59Q)), xvii) SEQ ID NO:73 (IL-12p40(D161N)), xviii)SEQ ID NO:74 (IL-12p40(E73Q)), xix) SEQ ID NO:75 (IL-12p40(Q144E)), xx)SEQ ID NO:76 (IL-12p40(E262Q)), xxi) SEQ ID NO:77 (IL-12p40(E100Q)),xxii) SEQ ID NO:78 (IL-12p40(D18N)), xxiii) SEQ ID NO:79(IL-12p40(E33Q)), xxiv) SEQ ID NO:80 (IL-12p40(Q229E)), xxv) SEQ IDNO:81 (IL-12p40(E235Q)), xxvi) SEQ ID NO:82 (IL-12p40(Q256N)), xxvii)SEQ ID NO:83 (IL-12p40(E299Q)), xxviii) SEQ ID NO:84 (IL-12p40(D87N)),xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii)IL-12p40(S43K), xxxiii) SEQ ID NO:379 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E)), xxxiv) SEQ ID NO:205(IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii)IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl)SEQ ID NO:206 (IL-12p40(E32Q/E59Q)), xli) SEQ ID NO:207(IL-12p40(D34N/E59Q)), xlii) SEQ ID NO:208 (IL-12p40(E59Q/E187Q)),xliii) SEQ ID NO:209 (IL-12p40(S43E/E59Q)), xliv) SEQ ID NO:210(IL-12p40(S43K/E49Q)), xlv) SEQ ID NO:211 (IL-12p40(E59Q/K163E)), xlvi)SEQ ID NO:212 (IL-12p40(E59Q/K99E)), xlvii) SEQ ID NO:213(IL-12p40(E59Q/K258E)), xlviii) SEQ ID NO:214 (IL-12p40(E59Q/K260E)),xlix) SEQ ID NO: 326 (IL-12p40 (D34N/E59K)), 1) SEQ ID NO: 325(IL-12p40(E59K/K99E)), li) SEQ ID NO: 339 (IL-12p40(D18K/E59K/K99E)),lii) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), liii) SEQ ID NO: 336(IL-12p40 (E59K/K99Y)), liv) SEQ ID NO: 335 (IL-12p40 (E59Y/K99E)), lv)SEQ ID NO: 338 (IL-12p40 (E45K/E59K/K99E)), lvi) SEQ ID NO: 340(IL-12p40 (E59K/K99E/Q144E)), lvii) SEQ ID NO: 341 (IL-12p40(E59K/K99E/Q144K)), lviii) SEQ ID NO: 342 (IL-12p40 (E59K/K99E/R159E)),lix) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), lx) SEQ ID NO: 344(IL-12p40 (D18K/E59K/K99E/K264E)), lxi) SEQ ID NO: 360 (IL-12p40(C252S)), lxii) SEQ ID NO: 361 (IL-12p40 (DI8K/E59K/K99E/C252S)), lxiii)SEQ ID NO: 362 (IL-12p40 (D18K/E59K/K99E/C252S/K264E)), lxiv) SEQ ID NO:363 (IL-12p40 (E59K/K99Y/C252S)), lxv) SEQ ID NO: 364 (IL-12p40(E59K/K99E/C252S/K264E)), lxvi) SEQ ID NO: 365 (IL-12p40(E59K/K99E/C252S)), lxvii) SEQ ID NO: 254 (IL-12p40 (N103D/N113D)),lxviii) SEQ ID NO: 255 (IL-12p40 (N103D/N200D)), lxix) SEQ ID NO: 256(IL-12p40 (N103D/N281D)), lxx) SEQ ID NO: 257 (IL-12p40 (N113D/N200D)),lxxi) SEQ ID NO: 258 (IL-12p40 (N113D/N281D)), lxxii) SEQ ID NO: 259(IL-12p40 (N200D/N281D)), lxxiii) SEQ ID NO: 260 (IL-12p40(N103D/N113D/N200D)), lxxiv) SEQ ID NO: 261 (IL-12p40(N103D/N113D/N281D)), lxxv) SEQ ID NO: 262 (IL-12p40(N103D/N200D/N281D)), lxxvi) SEQ ID NO: 263 (IL-12p40(N113D/N200D/N281D)), lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)),lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)), lxxix) SEQ ID NO: 266(IL-12p40 (N200Q)), lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)), lxxxi) SEQID NO: 268 (IL-12p40 (N103Q/N113Q)), lxxxii) SEQ ID NO: 269 (IL-12p40(N103Q/N200Q)), lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q/N281Q)),lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q/N200Q)), lxxxv) SEQ ID NO: 272(IL-12p40 (N113Q/N281Q)), lxxxvi) SEQ ID NO: 273 (IL-12p40(N200Q/N281Q)), lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q/N113Q/N200Q)),lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q/N113Q/N281Q)), lxxxix) SEQ IDNO: 276 (IL-12p40 (N103Q/N200Q/N281Q)), xc) SEQ ID NO: 277 (IL-12p40(N113Q/N200Q/N281Q)), xci) SEQ ID NO:278 (IL-12p40(N103Q/N113Q/N200Q/N281Q)), xcii) SEQ ID NO:327 (IL-12p40(D34N/E59K/K99E)), xciii) SEQ ID NO:328 (IL-12p40 (D34K/E59K/K99E)),xciv) SEQ ID NO:329 (IL-12p40 (E32Q/D34N/E59K/K99E)), xcv) SEQ ID NO:331(IL-12p40 (E32K/D34N/E59K/K99E)), xcvi) SEQ ID NO: 337 (IL-12p40(E59Y/K99Y)), xcvii) SEQ ID NO:366 (IL-12p40(E59K/K99E/N103Q/C252S/K264E)), xcviii) SEQ ID NO:367 (IL-12p40(E59K/K99E/N113Q/C252S/K264E)), xcix) SEQ ID NO:368 (IL-12p40(E59K/K99E/N200Q/C252S/K264E)), c) SEQ ID NO:369 (IL-12p40(E59K/K99E/N281Q/C252S/K264E)), ci) SEQ ID NO:370 (IL-12p40(E59K/K99E/N103Q/N113Q/C252S/K264E)), cii) SEQ ID NO:371 (IL-12p40(E59K/K99E/N103Q/N200Q/C252S/K264E)), ciii) SEQ ID NO:372 (IL-12p40(E59K/K99E/N103Q/N281Q/C252S/K264E)), civ) SEQ ID NO:373 (IL-12p40(E59K/K99E/N113Q/N200Q/C252S/K264E)), cv) SEQ ID NO:374 (IL-12p40(E59K/K99E/N113Q/N281Q/C252S/K264E)), cvi) SEQ ID NO:375 (IL-12p40(E59K/K99E/N200Q/N281Q/C252S/K264E)), cvii) SEQ ID NO:376 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E)), cviii) SEQ ID NO:377(IL-12p40 (E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E)), and cix) SEQ IDNO:378 (IL-12p40 (E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E)). In someembodiments, the present invention provides a heterodimeric Fc fusionprotein wherein said IL-12p35 subunit has one or more amino acidmodifications as amino acid residues selected from the group consistingof Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63,L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97,L124, M125, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163,D165, I171, R181, 1182, R183, V185, T186, D188, R189, V190, S192, Y193,N195, and A196.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidsubstitutions selected from the group consisting of N21D, Q35D, E38Q,D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A,L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K,E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p35 subunit has amino acidsubstitutions selected from the group consisting of N71D/N85D/N195D,N151D/E153Q, N151D/D165N, Q130E/N151D, N151D/K158E, E79Q/N151D,D55Q/N151D, N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q, N71Q/N195Q,N85Q/N195Q, N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, and N85D/N195D.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p35 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:113(IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115(IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D/N85D/N195D)), v) SEQID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii)SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(Q135E)),ix) SEQ ID NO:121 (IL-12p35(Q35D)), x) SEQ ID NO:122 (IL-12p35(Q146E)),xi) SEQ ID NO:123 (IL-12p35(N76D)), xii) SEQ ID NO:124(IL-12p35(E162Q)), xiii) SEQ ID NO:125 (IL-12p35(E163Q)), xiv)IL-12p35(N21D), xv) SEQ ID NO:333 (IL-12p35(D55Q)), xvi) IL-12p35(E79Q),xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx)SEQ ID NO:227 (IL-12p35(N151K)), xxi) SEQ ID NO:226 (IL-12p35(E153K)),xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO:225(IL-12p35(N151D/E153Q)), xxv) SEQ ID NO:228 (IL-12p35(N151D/D165N)),xxvi) SEQ ID NO:229 (IL-12p35(Q130E/N151D)), xxvii) SEQ ID NO:230(IL-12p35(N151D/K158E)), xxviii) SEQ ID NO:231 (IL-12p35(E79Q/N151D)),xxix) SEQ ID NO:232 (IL-12p35(D55Q/N151D)), xxx) SEQ ID NO:233(IL-12p35(N136D/N151D)), xxxi) SEQ ID NO:234 (IL-12p35(N21D/N151D)),xxxii) SEQ ID NO:235 (IL-12p35(E143Q/N151D)), xxxiii) SEQ ID NO: 345(IL-12p35(F96A)), xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)), xxxv) SEQ IDNO: 347 (IL-12p35(L89A)), xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)),xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)), xxxviii) SEQ ID NO: 350(IL-12p35(L75A)), xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)), xxxv) SEQ IDNO: 279 (IL-12p35 (N71Q)), xxxvi) SEQ ID NO: 280 (IL-12p35 (N85Q)),xxxvii) SEQ ID NO: 281 (IL-12p35 (N195Q)), xxxviii) SEQ ID NO: 282(IL-12p35 (N71Q/N85Q)), xxxix) SEQ ID NO: 283 (IL-12p35 (N71Q/N195Q)),xl) SEQ ID NO: 284 (IL-12p35 (N85Q/N195Q), xli) SEQ ID NO: 285 (IL-12p35(N71Q/N85Q/N195Q)), xlii) SEQ ID NO: 286 (IL-12p35 (N71D/N85D)), xliii)SEQ ID NO: 287 (IL-12p35 (N71D/N195D), xliv) SEQ ID NO: 288 (IL-12p35(N85D/N195D)), xlv) SEQ ID NO: 333 (IL-12p35 (D55Q)), and xlvi) SEQ IDNO: 334 (IL-12p35 (D55K)).

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said heterodimeric Fc fusion protein comprises:a) said first fusion protein having a polypeptide sequence of SEQ IDNO:49 (XENP27202 Chain 1), and b) said second fusion protein having apolypeptide sequence of SEQ ID NO:50 (XENP27202 Chain 2).

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said heterodimeric Fc fusion protein isXENP27202.

In some embodiments, the present invention provides a compositioncomprising a heterodimeric Fc fusion protein for use in treating cancerin a subject.

In some embodiments, the present invention provides one or more nucleicacids encoding a heterodimeric Fc fusion protein.

In some embodiments, the present invention provides a host cellcomprising said one or more nucleic acids encoding a heterodimeric Fcfusion protein.

In some embodiments, the present invention provides a method of making aheterodimeric Fc fusion protein, said method comprising culturing a hostcell under conditions whereby said heterodimeric Fc fusion protein isproduced.

In some embodiments, the present invention provides a method ofpurifying a heterodimeric Fc fusion protein, said method comprising: a)providing a composition comprising the heterodimeric Fc fusion protein;b) loading said composition onto an ion exchange column; and c)collecting a fraction containing said heterodimeric Fc fusion protein.

In another aspect, the present invention provides a heterodimeric Fcfusion protein comprising: a) a fusion protein comprising a firstprotein domain, a second protein domain and a first Fc domain, whereinsaid first protein domain is covalently attached to said second proteindomain, and wherein said second protein domain is covalently attached tothe N-terminus of said first Fc domain; and b) a second Fc domain;wherein said first and said second Fc domains comprise modificationspromoting heterodimerization of said first and said second Fc domainsand wherein said first protein domain comprises an IL-12p40 subunit andsaid second protein domain comprises an IL-12p35 subunit.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said modifications promoting heterodimerizationof said first and said second Fc domains are a set of amino acidsubstitutions selected from the group consisting of L368D/K370S andS364K; L368D/K370S and S364K/E357L; L368D/K370S and S364K/E357Q;T411E/K360E/Q362E and D401K; L368E/K370S and S364K; K370S andS364K/E357Q and T366S/L368A/Y407V:T366W (optionally including a bridgingdisulfide, T366S/L368A/Y407V/Y349C:T366W/S354C), according to EUnumbering.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said first protein domain is attached to saidsecond protein domain using a first domain linker and/or said secondprotein domain is attached to said first Fc domain using a second domainlinker.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said first and/or said second Fc domains have anadditional set of amino acid substitutions comprisingQ295E/N384D/Q418E/N421D, according to EU numbering.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said first and/or said second Fc domains have anadditional set of amino acid substitutions selected from the groupconsisting of G236R/L328R, E233P/L234V/L235A/G236_/S239K,E233P/L234V/L235A/G236_/S239K/A327G,E233P/L234V/L235A/G236_/S267K/A327G, E233P/L234V/L235A/G236_, andE233P/L234V/L235A/G236_/S267K, according to EU numbering.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected form the group consisting of SEQ ID NO:3 (human IL-12 subunitbeta (IL-12p40) precursor sequence) and SEQ ID NO:4 (human IL-12 subunitbeta (IL-12p40) mature form sequence), and/or said IL-12p35 subunit hasa polypeptide sequence selected from the group consisting of SEQ ID NO:1(human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ IDNO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said first and second Fc domains further compriseamino acid substitutions M428L/N434S.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit and/or said IL-12p35 subunit is a variant IL-12p35 subunit.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex; and/or said IL-12p35 subunit is a variant IL-12p35subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidmodifications at amino acid residues selected from the group consistingof E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32,E33, D34, L40, D41, Q42, S43, E45, L47, T54, 155, Q56, K58, E59, F60,G61, D62, Q65, Y66, E73, K84, E86, D87, G88, 189, W90, D93, D97, K99,E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156,R159, D161, N162, K163, D166, D170, Q172, D174, A176, C177, P178, A179,A180, E181, S183, P185, E187, N200, S204, F206, R208, D209, D214, N218,Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F247, S248, Q256,K158, C252, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292,Y293, and E299.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidsubstitutions selected from the group consisting of D18N, D18K, E32Q,E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q,D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N,R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N,K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has amino acidsubstitutions selected from the group consisting ofN103D/N113D/N200D/N281D, Q42E/E45Q, E45Q/Q56E, Q42E/E59Q, Q56E/E59Q,Q42E/E45Q/Q56E, E45Q/Q56E/E59Q, E32Q/E59Q, D34N/E59K, D34N/E59K/K99E,D34K/E59K/K99E, E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q,E59Q/E187Q, S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E,E59Q/K260E, E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y,E59Y/K99Y, E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:57(IL-12p40(N103D)), ii) SEQ ID NO:58 (IL-12p40(N113D)), iii) SEQ ID NO:59(IL-12p40(N200D)), iv) SEQ ID NO:60 (IL-12p40(N281D)), v) SEQ ID NO:61(IL-12p40(N103D/N113D/N200D/N281D)), vi) SEQ ID NO:62 (IL-12p40(Q42E)),vii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)),ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi)SEQ ID NO:67 (IL-12p40(Q42E/E45Q)), xii) SEQ ID NO:68(IL-12p40(E45Q/Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E/E59Q)), xiv)SEQ ID NO:70 (IL-12p40(Q56E/E59Q)), xv) SEQ ID NO:71(IL-12p40(Q42E/E45Q/Q56E)), xvi) SEQ ID NO:72(IL-12p40(E45Q/Q56E/E59Q)), xvii) SEQ ID NO:73 (IL-12p40(D161N)), xviii)SEQ ID NO:74 (IL-12p40(E73Q)), xix) SEQ ID NO:75 (IL-12p40(Q144E)), xx)SEQ ID NO:76 (IL-12p40(E262Q)), xxi) SEQ ID NO:77 (IL-12p40(E100Q)),xxii) SEQ ID NO:78 (IL-12p40(D18N)), xxiii) SEQ ID NO:79(IL-12p40(E33Q)), xxiv) SEQ ID NO:80 (IL-12p40(Q229E)), xxv) SEQ IDNO:81 (IL-12p40(E235Q)), xxvi) SEQ ID NO:82 (IL-12p40(Q256N)), xxvii)SEQ ID NO:83 (IL-12p40(E299Q)), xxviii) SEQ ID NO:84 (IL-12p40(D87N)),xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii)IL-12p40(S43K), xxxiii) SEQ ID NO:379 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E)), xxxiv) SEQ ID NO:205(IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii)IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl)SEQ ID NO:206 (IL-12p40(E32Q/E59Q)), xli) SEQ ID NO:207(IL-12p40(D34N/E59Q)), xlii) SEQ ID NO:208 (IL-12p40(E59Q/E187Q)),xliii) SEQ ID NO:209 (IL-12p40(S43E/E59Q)), xliv) SEQ ID NO:210(IL-12p40(S43K/E49Q)), xlv) SEQ ID NO:211 (IL-12p40(E59Q/K163E)), xlvi)SEQ ID NO:212 (IL-12p40(E59Q/K99E)), xlvii) SEQ ID NO:213(IL-12p40(E59Q/K258E)), xlviii) SEQ ID NO:214 (IL-12p40(E59Q/K260E)),xlix) SEQ ID NO: 326 (IL-12p40 (D34N/E59K)), 1) SEQ ID NO: 325(IL-12p40(E59K/K99E)), li) SEQ ID NO: 339 (IL-12p40(D18K/E59K/K99E)),lii) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), liii) SEQ ID NO: 336(IL-12p40 (E59K/K99Y)), liv) SEQ ID NO: 335 (IL-12p40 (E59Y/K99E)), lv)SEQ ID NO: 338 (IL-12p40 (E45K/E59K/K99E)), lvi) SEQ ID NO: 340(IL-12p40 (E59K/K99E/Q144E)), lvii) SEQ ID NO: 341 (IL-12p40(E59K/K99E/Q144K)), lviii) SEQ ID NO: 342 (IL-12p40 (E59K/K99E/R159E)),lix) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), lx) SEQ ID NO: 344(IL-12p40 (D18K/E59K/K99E/K264E)), lxi) SEQ ID NO: 360 (IL-12p40(C252S)), lxii) SEQ ID NO: 361 (IL-12p40 (DI8K/E59K/K99E/C252S)), lxiii)SEQ ID NO: 362 (IL-12p40 (D18K/E59K/K99E/C252S/K264E)), lxiv) SEQ ID NO:363 (IL-12p40 (E59K/K99Y/C252S)), lxv) SEQ ID NO: 364 (IL-12p40(E59K/K99E/C252S/K264E)), lxvi) SEQ ID NO: 365 (IL-12p40(E59K/K99E/C252S)), lxvii) SEQ ID NO: 254 (IL-12p40 (N103D/N113D)),lxviii) SEQ ID NO: 255 (IL-12p40 (N103D/N200D)), lxix) SEQ ID NO: 256(IL-12p40 (N103D/N281D)), lxx) SEQ ID NO: 257 (IL-12p40 (N113D/N200D)),lxxi) SEQ ID NO: 258 (IL-12p40 (N113D/N281D)), lxxii) SEQ ID NO: 259(IL-12p40 (N200D/N281D)), lxxiii) SEQ ID NO: 260 (IL-12p40(N103D/N113D/N200D)), lxxiv) SEQ ID NO: 261 (IL-12p40(N103D/N113D/N281D)), lxxv) SEQ ID NO: 262 (IL-12p40(N103D/N200D/N281D)), lxxvi) SEQ ID NO: 263 (IL-12p40(N113D/N200D/N281D)), lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)),lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)), lxxix) SEQ ID NO: 266(IL-12p40 (N200Q)), lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)), lxxxi) SEQID NO: 268 (IL-12p40 (N103Q/N113Q)), lxxxii) SEQ ID NO: 269 (IL-12p40(N103Q/N200Q)), lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q/N281Q)),lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q/N200Q)), lxxxv) SEQ ID NO: 272(IL-12p40 (N113Q/N281Q)), lxxxvi) SEQ ID NO: 273 (IL-12p40(N200Q/N281Q)), lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q/N113Q/N200Q)),lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q/N113Q/N281Q)), lxxxix) SEQ IDNO: 276 (IL-12p40 (N103Q/N200Q/N281Q)), xc) SEQ ID NO: 277 (IL-12p40(N113Q/N200Q/N281Q)), xci) SEQ ID NO:278 (IL-12p40(N103Q/N113Q/N200Q/N281Q)), xcii) SEQ ID NO:327 (IL-12p40(D34N/E59K/K99E)), xciii) SEQ ID NO:328 (IL-12p40 (D34K/E59K/K99E)),xciv) SEQ ID NO:329 (IL-12p40 (E32Q/D34N/E59K/K99E)), xcv) SEQ ID NO:331(IL-12p40 (E32K/D34N/E59K/K99E)), xcvi) SEQ ID NO: 337 (IL-12p40(E59Y/K99Y)), xcvii) SEQ ID NO:366 (IL-12p40(E59K/K99E/N103Q/C252S/K264E)), xcviii) SEQ ID NO:367 (IL-12p40(E59K/K99E/N113Q/C252S/K264E)), xcix) SEQ ID NO:368 (IL-12p40(E59K/K99E/N200Q/C252S/K264E)), c) SEQ ID NO:369 (IL-12p40(E59K/K99E/N281Q/C252S/K264E)), ci) SEQ ID NO:370 (IL-12p40(E59K/K99E/N103Q/N113Q/C252S/K264E)), cii) SEQ ID NO:371 (IL-12p40(E59K/K99E/N103Q/N200Q/C252S/K264E)), ciii) SEQ ID NO:372 (IL-12p40(E59K/K99E/N103Q/N281Q/C252S/K264E)), civ) SEQ ID NO:373 (IL-12p40(E59K/K99E/N113Q/N200Q/C252S/K264E)), cv) SEQ ID NO:374 (IL-12p40(E59K/K99E/N113Q/N281Q/C252S/K264E)), cvi) SEQ ID NO:375 (IL-12p40(E59K/K99E/N200Q/N281Q/C252S/K264E)), cvii) SEQ ID NO:376 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E)), cviii) SEQ ID NO:377(IL-12p40 (E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E)), and cix) SEQ IDNO:378 (IL-12p40 (E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E)).

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidmodifications as amino acid residues selected from the group consistingof Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63,L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97,L124, M125, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163,D165, I171, R181, 1182, R183, V185, T186, D188, R189, V190, S192, Y193,N195, and A196.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidsubstitutions selected from the group consisting of N21D, Q35D, E38Q,D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A,L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K,E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p35 subunit has amino acidsubstitutions selected from the group consisting of N71D/N85D/N195D,N151D/E153Q, N151D/D165N, Q130E/N151D, N151D/K158E, E79Q/N151D,D55Q/N151D, N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q, N71Q/N195Q,N85Q/N195Q, N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, and N85D/N195D.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p35 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:113(IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115(IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D/N85D/N195D)), v) SEQID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii)SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(Q135E)),ix) SEQ ID NO:121 (IL-12p35(Q35D)), x) SEQ ID NO:122 (IL-12p35(Q146E)),xi) SEQ ID NO:123 (IL-12p35(N76D)), xii) SEQ ID NO:124(IL-12p35(E162Q)), xiii) SEQ ID NO:125 (IL-12p35(E163Q)), xiv)IL-12p35(N21D), xv) SEQ ID NO:333 (IL-12p35(D55Q)), xvi) IL-12p35(E79Q),xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx)SEQ ID NO:227 (IL-12p35(N151K)), xxi) SEQ ID NO:226 (IL-12p35(E153K)),xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO:225(IL-12p35(N151D/E153Q)), xxv) SEQ ID NO:228 (IL-12p35(N151D/D165N)),xxvi) SEQ ID NO:229 (IL-12p35(Q130E/N151D)), xxvii) SEQ ID NO:230(IL-12p35(N151D/K158E)), xxviii) SEQ ID NO:231 (IL-12p35(E79Q/N151D)),xxix) SEQ ID NO:232 (IL-12p35(D55Q/N151D)), xxx) SEQ ID NO:233(IL-12p35(N136D/N151D)), xxxi) SEQ ID NO:234 (IL-12p35(N21D/N151D)),xxxii) SEQ ID NO:235 (IL-12p35(E143Q/N151D)), xxxiii) SEQ ID NO: 345(IL-12p35(F96A)), xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)), xxxv) SEQ IDNO: 347 (IL-12p35(L89A)), xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)),xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)), xxxviii) SEQ ID NO: 350(IL-12p35(L75A)), xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)), xxxv) SEQ IDNO: 279 (IL-12p35 (N71Q)), xxxvi) SEQ ID NO: 280 (IL-12p35 (N85Q)),xxxvii) SEQ ID NO: 281 (IL-12p35 (N195Q)), xxxviii) SEQ ID NO: 282(IL-12p35 (N71Q/N85Q)), xxxix) SEQ ID NO: 283 (IL-12p35 (N71Q/N195Q)),xl) SEQ ID NO: 284 (IL-12p35 (N85Q/N195Q), xli) SEQ ID NO: 285 (IL-12p35(N71Q/N85Q/N195Q)), xlii) SEQ ID NO: 286 (IL-12p35 (N71D/N85D)), xliii)SEQ ID NO: 287 (IL-12p35 (N71D/N195D), xliv) SEQ ID NO: 288 (IL-12p35(N85D/N195D)), xlv) SEQ ID NO: 333 (IL-12p35 (D55Q)), and xlvi) SEQ IDNO: 334 (IL-12p35 (D55K)).

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said heterodimeric Fc fusion protein comprises:a) said fusion protein having a polypeptide sequence of SEQ ID NO:51(XENP27203 Chain 1), and b) said second Fc domain having a polypeptidesequence of SEQ ID NO:52 (XENP27203 Chain 2).

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said heterodimeric Fc fusion protein isXENP27203.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said heterodimeric Fc fusion protein isXENP31290.

In some embodiments, the present invention provides a compositioncomprising a heterodimeric Fc fusion protein for use in treating cancerin a subject.

In some embodiments, the present invention provides one or more nucleicacids encoding a heterodimeric Fc fusion protein.

In some embodiments, the present invention provides a host cellcomprising said one or more nucleic acids encoding a heterodimeric Fcfusion protein.

In some embodiments, the present invention provides a method of making aheterodimeric Fc fusion protein, said method comprising culturing a hostcell under conditions whereby said heterodimeric Fc fusion protein isproduced.

In some embodiments, the present invention provides a method ofpurifying a heterodimeric Fc fusion protein, said method comprising: a)providing a composition comprising the heterodimeric Fc fusion protein;b) loading said composition onto an ion exchange column; and c)collecting a fraction containing said heterodimeric Fc fusion protein.

In another aspect, the present invention provides a heterodimeric Fcfusion protein comprising: a) a fusion protein comprising a firstprotein domain, a second protein domain and a first Fc domain, whereinsaid first protein domain is covalently attached to said second proteindomain, and wherein said second protein domain is covalently attached tothe N-terminus of said first Fc domain; and b) a second Fc domain;wherein said first and said second Fc domains comprise modificationspromoting heterodimerization of said first and said second Fc domainsand wherein said first protein domain comprises an IL-12p35 subunit andsaid second protein domain comprises an IL-12p40 subunit.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said modifications promoting heterodimerizationof said first and said second Fc domains are a set of amino acidsubstitutions selected from the group consisting of L368D/K370S andS364K; L368D/K370S and S364K/E357L; L368D/K370S and S364K/E357Q;T411E/K360E/Q362E and D401K; L368E/K370S and S364K; K370S andS364K/E357Q and T366S/L368A/Y407V:T366W (optionally including a bridgingdisulfide, T366S/L368A/Y407V/Y349C:T366W/S354C), according to EUnumbering.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said first protein domain is attached to saidsecond protein domain using a first domain linker and/or said secondprotein domain is attached to said first Fc domain using a second domainlinker.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said first and/or said second Fc domains have anadditional set of amino acid substitutions comprisingQ295E/N384D/Q418E/N421D, according to EU numbering.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said first and/or said second Fc domains have anadditional set of amino acid substitutions selected from the groupconsisting of G236R/L328R, E233P/L234V/L235A/G236_/S239K,E233P/L234V/L235A/G236_/S239K/A327G,E233P/L234V/L235A/G236_/S267K/A327G, E233P/L234V/L235A/G236_, andE233P/L234V/L235A/G236_/S267K, according to EU numbering.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected form the group consisting of SEQ ID NO:3 (human IL-12 subunitbeta (IL-12p40) precursor sequence) and/or SEQ ID NO:4 (human IL-12subunit beta (IL-12p40) mature form sequence), and said IL-12p35 subunithas a polypeptide sequence selected from the group consisting of SEQ IDNO:1 (human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQID NO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said first and second Fc domains further compriseamino acid substitutions M428L/N434S.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit and/or said IL-12p35 subunit is a variant IL-12p35 subunit.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex; and/or said IL-12p35 subunit is a variant IL-12p35subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidmodifications at amino acid residues selected from the group consistingof E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32,E33, D34, L40, D41, Q42, S43, E45, L47, T54, 155, Q56, K58, E59, F60,G61, D62, Q65, Y66, E73, K84, E86, D87, G88, 189, W90, D93, D97, K99,E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156,R159, D161, N162, K163, D166, D170, Q172, D174, A176, C177, P178, A179,A180, E181, S183, P185, E187, N200, S204, F206, R208, D209, D214, N218,Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F247, S248, C252,Q256, K258, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292,Y293, and E299.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidsubstitutions selected from the group consisting of D18N, D18K, E32Q,E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q,D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N,R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N,K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has amino acidsubstitutions selected from the group consisting ofN103D/N113D/N200D/N281D, Q42E/E45Q, E45Q/Q56E, Q42E/E59Q, Q56E/E59Q,Q42E/E45Q/Q56E, E45Q/Q56E/E59Q, E32Q/E59Q, D34N/E59K, D34N/E59K/K99E,D34K/E59K/K99E, E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q,E59Q/E187Q, S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E,E59Q/K260E, E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y,E59Y/K99Y, E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:57(IL-12p40(N103D)), ii) SEQ ID NO:58 (IL-12p40(N113D)), iii) SEQ ID NO:59(IL-12p40(N200D)), iv) SEQ ID NO:60 (IL-12p40(N281D)), v) SEQ ID NO:61(IL-12p40(N103D/N113D/N200D/N281D)), vi) SEQ ID NO:62 (IL-12p40(Q42E)),vii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)),ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi)SEQ ID NO:67 (IL-12p40(Q42E/E45Q)), xii) SEQ ID NO:68(IL-12p40(E45Q/Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E/E59Q)), xiv)SEQ ID NO:70 (IL-12p40(Q56E/E59Q)), xv) SEQ ID NO:71(IL-12p40(Q42E/E45Q/Q56E)), xvi) SEQ ID NO:72(IL-12p40(E45Q/Q56E/E59Q)), xvii) SEQ ID NO:73 (IL-12p40(D161N)), xviii)SEQ ID NO:74 (IL-12p40(E73Q)), xix) SEQ ID NO:75 (IL-12p40(Q144E)), xx)SEQ ID NO:76 (IL-12p40(E262Q)), xxi) SEQ ID NO:77 (IL-12p40(E100Q)),xxii) SEQ ID NO:78 (IL-12p40(D18N)), xxiii) SEQ ID NO:79(IL-12p40(E33Q)), xxiv) SEQ ID NO:80 (IL-12p40(Q229E)), xxv) SEQ IDNO:81 (IL-12p40(E235Q)), xxvi) SEQ ID NO:82 (IL-12p40(Q256N)), xxvii)SEQ ID NO:83 (IL-12p40(E299Q)), xxviii) SEQ ID NO:84 (IL-12p40(D87N)),xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii)IL-12p40(S43K), xxxiii) SEQ ID NO:379 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E)), xxxiv) SEQ ID NO:205(IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii)IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl)SEQ ID NO:206 (IL-12p40(E32Q/E59Q)), xli) SEQ ID NO:207(IL-12p40(D34N/E59Q)), xlii) SEQ ID NO:208 (IL-12p40(E59Q/E187Q)),xliii) SEQ ID NO:209 (IL-12p40(S43E/E59Q)), xliv) SEQ ID NO:210(IL-12p40(S43K/E49Q)), xlv) SEQ ID NO:211 (IL-12p40(E59Q/K163E)), xlvi)SEQ ID NO:212 (IL-12p40(E59Q/K99E)), xlvii) SEQ ID NO:213(IL-12p40(E59Q/K258E)), xlviii) SEQ ID NO:214 (IL-12p40(E59Q/K260E)),xlix) SEQ ID NO: 326 (IL-12p40 (D34N/E59K)), 1) SEQ ID NO: 325(IL-12p40(E59K/K99E)), li) SEQ ID NO: 339 (IL-12p40(D18K/E59K/K99E)),lii) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), liii) SEQ ID NO: 336(IL-12p40 (E59K/K99Y)), liv) SEQ ID NO: 335 (IL-12p40 (E59Y/K99E)), lv)SEQ ID NO: 338 (IL-12p40 (E45K/E59K/K99E)), lvi) SEQ ID NO: 340(IL-12p40 (E59K/K99E/Q144E)), lvii) SEQ ID NO: 341 (IL-12p40(E59K/K99E/Q144K)), lviii) SEQ ID NO: 342 (IL-12p40 (E59K/K99E/R159E)),lix) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), lx) SEQ ID NO: 344(IL-12p40 (D18K/E59K/K99E/K264E)), lxi) SEQ ID NO: 360 (IL-12p40(C252S)), lxii) SEQ ID NO: 361 (IL-12p40 (DI8K/E59K/K99E/C252S)), lxiii)SEQ ID NO: 362 (IL-12p40 (D18K/E59K/K99E/C252S/K264E)), lxiv) SEQ ID NO:363 (IL-12p40 (E59K/K99Y/C252S)), lxv) SEQ ID NO: 364 (IL-12p40(E59K/K99E/C252S/K264E)), lxvi) SEQ ID NO: 365 (IL-12p40(E59K/K99E/C252S)), lxvii) SEQ ID NO: 254 (IL-12p40 (N103D/N113D)),lxviii) SEQ ID NO: 255 (IL-12p40 (N103D/N200D)), lxix) SEQ ID NO: 256(IL-12p40 (N103D/N281D)), lxx) SEQ ID NO: 257 (IL-12p40 (N113D/N200D)),lxxi) SEQ ID NO: 258 (IL-12p40 (N113D/N281D)), lxxii) SEQ ID NO: 259(IL-12p40 (N200D/N281D)), lxxiii) SEQ ID NO: 260 (IL-12p40(N103D/N113D/N200D)), lxxiv) SEQ ID NO: 261 (IL-12p40(N103D/N113D/N281D)), lxxv) SEQ ID NO: 262 (IL-12p40(N103D/N200D/N281D)), lxxvi) SEQ ID NO: 263 (IL-12p40(N113D/N200D/N281D)), lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)),lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)), lxxix) SEQ ID NO: 266(IL-12p40 (N200Q)), lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)), lxxxi) SEQID NO: 268 (IL-12p40 (N103Q/N113Q)), lxxxii) SEQ ID NO: 269 (IL-12p40(N103Q/N200Q)), lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q/N281Q)),lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q/N200Q)), lxxxv) SEQ ID NO: 272(IL-12p40 (N113Q/N281Q)), lxxxvi) SEQ ID NO: 273 (IL-12p40(N200Q/N281Q)), lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q/N113Q/N200Q)),lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q/N113Q/N281Q)), lxxxix) SEQ IDNO: 276 (IL-12p40 (N103Q/N200Q/N281Q)), xc) SEQ ID NO: 277 (IL-12p40(N113Q/N200Q/N281Q)), xci) SEQ ID NO:278 (IL-12p40(N103Q/N113Q/N200Q/N281Q)), xcii) SEQ ID NO:327 (IL-12p40(D34N/E59K/K99E)), xciii) SEQ ID NO:328 (IL-12p40 (D34K/E59K/K99E)),xciv) SEQ ID NO:329 (IL-12p40 (E32Q/D34N/E59K/K99E)), xcv) SEQ ID NO:331(IL-12p40 (E32K/D34N/E59K/K99E)), xcvi) SEQ ID NO: 337 (IL-12p40(E59Y/K99Y)), xcvii) SEQ ID NO:366 (IL-12p40(E59K/K99E/N103Q/C252S/K264E)), xcviii) SEQ ID NO:367 (IL-12p40(E59K/K99E/N113Q/C252S/K264E)), xcix) SEQ ID NO:368 (IL-12p40(E59K/K99E/N200Q/C252S/K264E)), c) SEQ ID NO:369 (IL-12p40(E59K/K99E/N281Q/C252S/K264E)), ci) SEQ ID NO:370 (IL-12p40(E59K/K99E/N103Q/N113Q/C252S/K264E)), cii) SEQ ID NO:371 (IL-12p40(E59K/K99E/N103Q/N200Q/C252S/K264E)), ciii) SEQ ID NO:372 (IL-12p40(E59K/K99E/N103Q/N281Q/C252S/K264E)), civ) SEQ ID NO:373 (IL-12p40(E59K/K99E/N113Q/N200Q/C252S/K264E)), cv) SEQ ID NO:374 (IL-12p40(E59K/K99E/N113Q/N281Q/C252S/K264E)), cvi) SEQ ID NO:375 (IL-12p40(E59K/K99E/N200Q/N281Q/C252S/K264E)), cvii) SEQ ID NO:376 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E)), cviii) SEQ ID NO:377(IL-12p40 (E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E)), and cix) SEQ IDNO:378 (IL-12p40 (E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E)).

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidmodifications as amino acid residues selected from the group consistingof Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63,L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97,L124, M125, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163,D165, I171, R181, 1182, R183, V185, T186, D188, R189, V190, S192, Y193,N195, and A196.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidsubstitutions selected from the group consisting of N21D, Q35D, E38Q,D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A,L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K,E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p35 subunit has amino acidsubstitutions selected from the group consisting of N71D/N85D/N195D,N151D/E153Q, N151D/D165N, Q130E/N151D, N151D/K158E, E79Q/N151D,D55Q/N151D, N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q, N71Q/N195Q,N85Q/N195Q, N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, and N85D/N195D.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p35 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:113(IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115(IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D/N85D/N195D)), v) SEQID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii)SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(Q135E)),ix) SEQ ID NO:121 (IL-12p35(Q35D)), x) SEQ ID NO:122 (IL-12p35(Q146E)),xi) SEQ ID NO:123 (IL-12p35(N76D)), xii) SEQ ID NO:124(IL-12p35(E162Q)), xiii) SEQ ID NO:125 (IL-12p35(E163Q)), xiv)IL-12p35(N21D), xv) SEQ ID NO:333 (IL-12p35(D55Q)), xvi) IL-12p35(E79Q),xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx)SEQ ID NO:227 (IL-12p35(N151K)), xxi) SEQ ID NO:226 (IL-12p35(E153K)),xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO:225(IL-12p35(N151D/E153Q)), xxv) SEQ ID NO:228 (IL-12p35(N151D/D165N)),xxvi) SEQ ID NO:229 (IL-12p35(Q130E/N151D)), xxvii) SEQ ID NO:230(IL-12p35(N151D/K158E)), xxviii) SEQ ID NO:231 (IL-12p35(E79Q/N151D)),xxix) SEQ ID NO:232 (IL-12p35(D55Q/N151D)), xxx) SEQ ID NO:233(IL-12p35(N136D/N151D)), xxxi) SEQ ID NO:234 (IL-12p35(N21D/N151D)),xxxii) SEQ ID NO:235 (IL-12p35(E143Q/N151D)), xxxiii) SEQ ID NO: 345(IL-12p35(F96A)), xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)), xxxv) SEQ IDNO: 347 (IL-12p35(L89A)), xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)),xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)), xxxviii) SEQ ID NO: 350(IL-12p35(L75A)), xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)), xxxv) SEQ IDNO: 279 (IL-12p35 (N71Q)), xxxvi) SEQ ID NO: 280 (IL-12p35 (N85Q)),xxxvii) SEQ ID NO: 281 (IL-12p35 (N195Q)), xxxviii) SEQ ID NO: 282(IL-12p35 (N71Q/N85Q)), xxxix) SEQ ID NO: 283 (IL-12p35 (N71Q/N195Q)),xl) SEQ ID NO: 284 (IL-12p35 (N85Q/N195Q), xli) SEQ ID NO: 285 (IL-12p35(N71Q/N85Q/N195Q)), xlii) SEQ ID NO: 286 (IL-12p35 (N71D/N85D)), xliii)SEQ ID NO: 287 (IL-12p35 (N71D/N195D), xliv) SEQ ID NO: 288 (IL-12p35(N85D/N195D)), xlv) SEQ ID NO: 333 (IL-12p35 (D55Q)), and xlvi) SEQ IDNO: 334 (IL-12p35 (D55K)).

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said heterodimeric Fc fusion protein comprises:a) said fusion protein having a polypeptide sequence of SEQ ID NO:53(XENP27204 Chain 1), and b) said second Fc domain having a polypeptidesequence of SEQ ID NO:54 (XENP27204 Chain 2).

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said heterodimeric Fc fusion protein isXENP27204.

In some embodiments, the present invention provides a compositioncomprising a heterodimeric Fc fusion protein for use in treating cancerin a subject.

In some embodiments, the present invention provides one or more nucleicacids encoding a heterodimeric Fc fusion protein.

In some embodiments, the present invention provides a host cellcomprising said one or more nucleic acids encoding a heterodimeric Fcfusion protein.

In some embodiments, the present invention provides a method of making aheterodimeric Fc fusion protein, said method comprising culturing a hostcell under conditions whereby said heterodimeric Fc fusion protein isproduced.

In some embodiments, the present invention provides a method ofpurifying a heterodimeric Fc fusion protein, said method comprising: a)providing a composition comprising the heterodimeric Fc fusion protein;b) loading said composition onto an ion exchange column; and c)collecting a fraction containing said heterodimeric Fc fusion protein.

In another aspect, the present invention provides a heterodimeric Fcfusion protein comprising: a) a fusion protein comprising a firstprotein domain, a second protein domain and a first Fc domain, whereinsaid first protein domain is attached to the C-terminus of said first Fcdomain, and wherein said second protein domain is covalently attached tosaid first protein domain; and b) a second Fc domain; wherein said firstand said second Fc domains comprise modifications promotingheterodimerization of said first and said second Fc domains and whereinsaid first protein domain comprises an IL-12p40 subunit and said secondprotein domain comprises an IL-12p35 subunit.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said modifications promoting heterodimerizationof said first and said second Fc domains are a set of amino acidsubstitutions selected from the group consisting of L368D/K370S andS364K; L368D/K370S and S364K/E357L; L368D/K370S and S364K/E357Q;T411E/K360E/Q362E and D401K; L368E/K370S and S364K; K370S andS364K/E357Q and T366S/L368A/Y407V:T366W (optionally including a bridgingdisulfide, T366S/L368A/Y407V/Y349C:T366W/S354C), according to EUnumbering.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said first protein domain is attached to saidsecond protein domain using a first domain linker and/or said secondprotein domain is attached to said first Fc domain using a second domainlinker.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said first and/or said second Fc domains have anadditional set of amino acid substitutions comprisingQ295E/N384D/Q418E/N421D, according to EU numbering.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said first and/or said second Fc domains have anadditional set of amino acid substitutions selected from the groupconsisting of G236R/L328R, E233P/L234V/L235A/G236_/S239K,E233P/L234V/L235A/G236_/S239K/A327G,E233P/L234V/L235A/G236_/S267K/A327G, E233P/L234V/L235A/G236_, andE233P/L234V/L235A/G236_/S267K, according to EU numbering.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected form the group consisting of SEQ ID NO:3 (human IL-12 subunitbeta (IL-12p40) precursor sequence) and SEQ ID NO:4 (human IL-12 subunitbeta (IL-12p40) mature form sequence), and/or said IL-12p35 subunit hasa polypeptide sequence selected from the group consisting of SEQ ID NO:1(human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ IDNO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said first and second Fc domains further compriseamino acid substitutions M428L/N434S.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit and/or said IL-12p35 subunit is a variant IL-12p35 subunit.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex; and/or said IL-12p35 subunit is a variant IL-12p35subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidmodifications at amino acid residues selected from the group consistingof E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32,E33, D34, L40, D41, Q42, S43, E45, L47, T54, 155, Q56, K58, E59, F60,G61, D62, Q65, Y66, E73, K84, E86, D87, G88, 189, W90, D93, D97, K99,E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156,R159, D161, N162, K163, D166, D170, Q172, D174, A176, C177, P178, A179,A180, E181, S183, P185, E187, N200, S204, F206, R208, D209, D214, N218,Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F247, S248, C252,Q256, K258, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292,Y293, and E299.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidsubstitutions selected from the group consisting of D18N, D18K, E32Q,E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q,D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N,R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N,K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has amino acidsubstitutions selected from the group consisting ofN103D/N113D/N200D/N281D, Q42E/E45Q, E45Q/Q56E, Q42E/E59Q, Q56E/E59Q,Q42E/E45Q/Q56E, E45Q/Q56E/E59Q, E32Q/E59Q, D34N/E59K, D34N/E59K/K99E,D34K/E59K/K99E, E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q,E59Q/E187Q, S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E,E59Q/K260E, E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y,E59Y/K99Y, E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:57(IL-12p40(N103D)), ii) SEQ ID NO:58 (IL-12p40(N113D)), iii) SEQ ID NO:59(IL-12p40(N200D)), iv) SEQ ID NO:60 (IL-12p40(N281D)), v) SEQ ID NO:61(IL-12p40(N103D/N113D/N200D/N281D)), vi) SEQ ID NO:62 (IL-12p40(Q42E)),vii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)),ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi)SEQ ID NO:67 (IL-12p40(Q42E/E45Q)), xii) SEQ ID NO:68(IL-12p40(E45Q/Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E/E59Q)), xiv)SEQ ID NO:70 (IL-12p40(Q56E/E59Q)), xv) SEQ ID NO:71(IL-12p40(Q42E/E45Q/Q56E)), xvi) SEQ ID NO:72(IL-12p40(E45Q/Q56E/E59Q)), xvii) SEQ ID NO:73 (IL-12p40(D161N)), xviii)SEQ ID NO:74 (IL-12p40(E73Q)), xix) SEQ ID NO:75 (IL-12p40(Q144E)), xx)SEQ ID NO:76 (IL-12p40(E262Q)), xxi) SEQ ID NO:77 (IL-12p40(E100Q)),xxii) SEQ ID NO:78 (IL-12p40(D18N)), xxiii) SEQ ID NO:79(IL-12p40(E33Q)), xxiv) SEQ ID NO:80 (IL-12p40(Q229E)), xxv) SEQ IDNO:81 (IL-12p40(E235Q)), xxvi) SEQ ID NO:82 (IL-12p40(Q256N)), xxvii)SEQ ID NO:83 (IL-12p40(E299Q)), xxviii) SEQ ID NO:84 (IL-12p40(D87N)),xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii)IL-12p40(S43K), xxxiii) SEQ ID NO:379 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E)), xxxiv) SEQ ID NO:205(IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii)IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl)SEQ ID NO:206 (IL-12p40(E32Q/E59Q)), xli) SEQ ID NO:207(IL-12p40(D34N/E59Q)), xlii) SEQ ID NO:208 (IL-12p40(E59Q/E187Q)),xliii) SEQ ID NO:209 (IL-12p40(S43E/E59Q)), xliv) SEQ ID NO:210(IL-12p40(S43K/E49Q)), xlv) SEQ ID NO:211 (IL-12p40(E59Q/K163E)), xlvi)SEQ ID NO:212 (IL-12p40(E59Q/K99E)), xlvii) SEQ ID NO:213(IL-12p40(E59Q/K258E)), xlviii) SEQ ID NO:214 (IL-12p40(E59Q/K260E)),xlix) SEQ ID NO: 326 (IL-12p40 (D34N/E59K)), 1) SEQ ID NO: 325(IL-12p40(E59K/K99E)), li) SEQ ID NO: 339 (IL-12p40(D18K/E59K/K99E)),lii) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), liii) SEQ ID NO: 336(IL-12p40 (E59K/K99Y)), liv) SEQ ID NO: 335 (IL-12p40 (E59Y/K99E)), lv)SEQ ID NO: 338 (IL-12p40 (E45K/E59K/K99E)), lvi) SEQ ID NO: 340(IL-12p40 (E59K/K99E/Q144E)), lvii) SEQ ID NO: 341 (IL-12p40(E59K/K99E/Q144K)), lviii) SEQ ID NO: 342 (IL-12p40 (E59K/K99E/R159E)),lix) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), lx) SEQ ID NO: 344(IL-12p40 (D18K/E59K/K99E/K264E)), lxi) SEQ ID NO: 360 (IL-12p40(C252S)), lxii) SEQ ID NO: 361 (IL-12p40 (DI8K/E59K/K99E/C252S)), lxiii)SEQ ID NO: 362 (IL-12p40 (D18K/E59K/K99E/C252S/K264E)), lxiv) SEQ ID NO:363 (IL-12p40 (E59K/K99Y/C252S)), lxv) SEQ ID NO: 364 (IL-12p40(E59K/K99E/C252S/K264E)), lxvi) SEQ ID NO: 365 (IL-12p40(E59K/K99E/C252S)), lxvii) SEQ ID NO: 254 (IL-12p40 (N103D/N113D)),lxviii) SEQ ID NO: 255 (IL-12p40 (N103D/N200D)), lxix) SEQ ID NO: 256(IL-12p40 (N103D/N281D)), lxx) SEQ ID NO: 257 (IL-12p40 (N113D/N200D)),lxxi) SEQ ID NO: 258 (IL-12p40 (N113D/N281D)), lxxii) SEQ ID NO: 259(IL-12p40 (N200D/N281D)), lxxiii) SEQ ID NO: 260 (IL-12p40(N103D/N113D/N200D)), lxxiv) SEQ ID NO: 261 (IL-12p40(N103D/N113D/N281D)), lxxv) SEQ ID NO: 262 (IL-12p40(N103D/N200D/N281D)), lxxvi) SEQ ID NO: 263 (IL-12p40(N113D/N200D/N281D)), lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)),lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)), lxxix) SEQ ID NO: 266(IL-12p40 (N200Q)), lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)), lxxxi) SEQID NO: 268 (IL-12p40 (N103Q/N113Q)), lxxxii) SEQ ID NO: 269 (IL-12p40(N103Q/N200Q)), lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q/N281Q)),lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q/N200Q)), lxxxv) SEQ ID NO: 272(IL-12p40 (N113Q/N281Q)), lxxxvi) SEQ ID NO: 273 (IL-12p40(N200Q/N281Q)), lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q/N113Q/N200Q)),lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q/N113Q/N281Q)), lxxxix) SEQ IDNO: 276 (IL-12p40 (N103Q/N200Q/N281Q)), xc) SEQ ID NO: 277 (IL-12p40(N113Q/N200Q/N281Q)), xci) SEQ ID NO:278 (IL-12p40(N103Q/N113Q/N200Q/N281Q)), xcii) SEQ ID NO:327 (IL-12p40(D34N/E59K/K99E)), xciii) SEQ ID NO:328 (IL-12p40 (D34K/E59K/K99E)),xciv) SEQ ID NO:329 (IL-12p40 (E32Q/D34N/E59K/K99E)), xcv) SEQ ID NO:331(IL-12p40 (E32K/D34N/E59K/K99E)), xcvi) SEQ ID NO: 337 (IL-12p40(E59Y/K99Y)), xcvii) SEQ ID NO:366 (IL-12p40(E59K/K99E/N103Q/C252S/K264E)), xcviii) SEQ ID NO:367 (IL-12p40(E59K/K99E/N113Q/C252S/K264E)), xcix) SEQ ID NO:368 (IL-12p40(E59K/K99E/N200Q/C252S/K264E)), c) SEQ ID NO:369 (IL-12p40(E59K/K99E/N281Q/C252S/K264E)), ci) SEQ ID NO:370 (IL-12p40(E59K/K99E/N103Q/N113Q/C252S/K264E)), cii) SEQ ID NO:371 (IL-12p40(E59K/K99E/N103Q/N200Q/C252S/K264E)), ciii) SEQ ID NO:372 (IL-12p40(E59K/K99E/N103Q/N281Q/C252S/K264E)), civ) SEQ ID NO:373 (IL-12p40(E59K/K99E/N113Q/N200Q/C252S/K264E)), cv) SEQ ID NO:374 (IL-12p40(E59K/K99E/N113Q/N281Q/C252S/K264E)), cvi) SEQ ID NO:375 (IL-12p40(E59K/K99E/N200Q/N281Q/C252S/K264E)), cvii) SEQ ID NO:376 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E)), cviii) SEQ ID NO:377(IL-12p40 (E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E)), and cix) SEQ IDNO:378 (IL-12p40 (E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E)).

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidmodifications as amino acid residues selected from the group consistingof Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63,L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97,L124, M125, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163,D165, I171, R181, 1182, R183, V185, T186, D188, R189, V190, S192, Y193,N195, and A196.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidsubstitutions selected from the group consisting of N21D, Q35D, E38Q,D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A,L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K,E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p35 subunit has amino acidsubstitutions selected from the group consisting of N71D/N85D/N195D,N151D/E153Q, N151D/D165N, Q130E/N151D, N151D/K158E, E79Q/N151D,D55Q/N151D, N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q, N71Q/N195Q,N85Q/N195Q, N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, and N85D/N195D.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p35 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:113(IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115(IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D/N85D/N195D)), v) SEQID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii)SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(Q135E)),ix) SEQ ID NO:121 (IL-12p35(Q35D)), x) SEQ ID NO:122 (IL-12p35(Q146E)),xi) SEQ ID NO:123 (IL-12p35(N76D)), xii) SEQ ID NO:124(IL-12p35(E162Q)), xiii) SEQ ID NO:125 (IL-12p35(E163Q)), xiv)IL-12p35(N21D), xv) SEQ ID NO:333 (IL-12p35(D55Q)), xvi) IL-12p35(E79Q),xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx)SEQ ID NO:227 (IL-12p35(N151K)), xxi) SEQ ID NO:226 (IL-12p35(E153K)),xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO:225(IL-12p35(N151D/E153Q)), xxv) SEQ ID NO:228 (IL-12p35(N151D/D165N)),xxvi) SEQ ID NO:229 (IL-12p35(Q130E/N151D)), xxvii) SEQ ID NO:230(IL-12p35(N151D/K158E)), xxviii) SEQ ID NO:231 (IL-12p35(E79Q/N151D)),xxix) SEQ ID NO:232 (IL-12p35(D55Q/N151D)), xxx) SEQ ID NO:233(IL-12p35(N136D/N151D)), xxxi) SEQ ID NO:234 (IL-12p35(N21D/N151D)),xxxii) SEQ ID NO:235 (IL-12p35(E143Q/N151D)), xxxiii) SEQ ID NO: 345(IL-12p35(F96A)), xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)), xxxv) SEQ IDNO: 347 (IL-12p35(L89A)), xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)),xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)), xxxviii) SEQ ID NO: 350(IL-12p35(L75A)), xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)), xxxv) SEQ IDNO: 279 (IL-12p35 (N71Q)), xxxvi) SEQ ID NO: 280 (IL-12p35 (N85Q)),xxxvii) SEQ ID NO: 281 (IL-12p35 (N195Q)), xxxviii) SEQ ID NO: 282(IL-12p35 (N71Q/N85Q)), xxxix) SEQ ID NO: 283 (IL-12p35 (N71Q/N195Q)),xl) SEQ ID NO: 284 (IL-12p35 (N85Q/N195Q), xli) SEQ ID NO: 285 (IL-12p35(N71Q/N85Q/N195Q)), xlii) SEQ ID NO: 286 (IL-12p35 (N71D/N85D)), xliii)SEQ ID NO: 287 (IL-12p35 (N71D/N195D), xliv) SEQ ID NO: 288 (IL-12p35(N85D/N195D)), xlv) SEQ ID NO: 333 (IL-12p35 (D55Q)), and xlvi) SEQ IDNO: 334 (IL-12p35 (D55K)).

In some embodiments, the present invention provides a compositioncomprising a heterodimeric Fc fusion protein for use in treating cancerin a subject.

In some embodiments, the present invention provides one or more nucleicacids encoding a heterodimeric Fc fusion protein.

In some embodiments, the present invention provides a host cellcomprising said one or more nucleic acids encoding a heterodimeric Fcfusion protein.

In some embodiments, the present invention provides a method of making aheterodimeric Fc fusion protein, said method comprising culturing a hostcell under conditions whereby said heterodimeric Fc fusion protein isproduced.

In some embodiments, the present invention provides a method ofpurifying a heterodimeric Fc fusion protein, said method comprising: a)providing a composition comprising the heterodimeric Fc fusion protein;b) loading said composition onto an ion exchange column; and c)collecting a fraction containing said heterodimeric Fc fusion protein.

In another aspect, the present invention provides a heterodimeric Fcfusion protein comprising: a) a fusion protein comprising a firstprotein domain, a second protein domain and a first Fc domain, whereinsaid first protein domain is attached to the C-terminus of said first Fcdomain, and wherein said second protein domain is covalently attached tosaid first protein domain; and b) a second Fc domain; wherein said firstand said second Fc domains comprise modifications promotingheterodimerization of said first and said second Fc domains and whereinsaid first protein domain comprises an IL-12p35 subunit and said secondprotein domain comprises an IL-12p40 subunit.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein, wherein said modifications promoting heterodimerizationof said first and said second Fc domains are a set of amino acidsubstitutions selected from the group consisting of L368D/K370S andS364K; L368D/K370S and S364K/E357L; L368D/K370S and S364K/E357Q;T411E/K360E/Q362E and D401K; L368E/K370S and S364K; K370S andS364K/E357Q and T366S/L368A/Y407V:T366W (optionally including a bridgingdisulfide, T366S/L368A/Y407V/Y349C:T366W/S354C), according to EUnumbering.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said first protein domain is attached to saidsecond protein domain using a first domain linker and/or said secondprotein domain is attached to said first Fc domain using a second domainlinker.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said first and/or said second Fc domains have anadditional set of amino acid substitutions comprisingQ295E/N384D/Q418E/N421D, according to EU numbering.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said first and/or said second Fc domains have anadditional set of amino acid substitutions selected from the groupconsisting of G236R/L328R, E233P/L234V/L235A/G236_/S239K,E233P/L234V/L235A/G236_/S239K/A327G,E233P/L234V/L235A/G236_/S267K/A327G, E233P/L234V/L235A/G236_, andE233P/L234V/L235A/G236_/S267K, according to EU numbering.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected form the group consisting of SEQ ID NO:3 (human IL-12 subunitbeta (IL-12p40) precursor sequence) and SEQ ID NO:4 (human IL-12 subunitbeta (IL-12p40) mature form sequence), and/or said IL-12p35 subunit hasa polypeptide sequence selected from the group consisting of SEQ ID NO:1(human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ IDNO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said first and second Fc domains further compriseamino acid substitutions M428L/N434S.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit and/or said IL-12p35 subunit is a variant IL-12p35 subunit.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex; and/or said IL-12p35 subunit is a variant IL-12p35subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidmodifications at amino acid residues selected from the group consistingof E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32,E33, D34, L40, D41, Q42, S43, E45, L47, T54, 155, Q56, K58, E59, F60,G61, D62, Q65, Y66, E73, K84, E86, D87, G88, 189, W90, D93, D97, K99,E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156,R159, D161, N162, K163, D166, D170, Q172, D174, A176, C177, P178, A179,A180, E181, S183, P185, E187, N200, S204, F206, R208, D209, D214, N218,Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F247, S248, C252,Q256, K258, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292,Y293, and E299.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidsubstitutions selected from the group consisting of D18N, D18K, E32Q,E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q,D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N,R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N,K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has amino acidsubstitutions selected from the group consisting ofN103D/N113D/N200D/N281D, Q42E/E45Q, E45Q/Q56E, Q42E/E59Q, Q56E/E59Q,Q42E/E45Q/Q56E, E45Q/Q56E/E59Q, E32Q/E59Q, D34N/E59K, D34N/E59K/K99E,D34K/E59K/K99E, E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q,E59Q/E187Q, S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E,E59Q/K260E, E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y,E59Y/K99Y, E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:57(IL-12p40(N103D)), ii) SEQ ID NO:58 (IL-12p40(N113D)), iii) SEQ ID NO:59(IL-12p40(N200D)), iv) SEQ ID NO:60 (IL-12p40(N281D)), v) SEQ ID NO:61(IL-12p40(N103D/N113D/N200D/N281D)), vi) SEQ ID NO:62 (IL-12p40(Q42E)),vii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)),ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi)SEQ ID NO:67 (IL-12p40(Q42E/E45Q)), xii) SEQ ID NO:68(IL-12p40(E45Q/Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E/E59Q)), xiv)SEQ ID NO:70 (IL-12p40(Q56E/E59Q)), xv) SEQ ID NO:71(IL-12p40(Q42E/E45Q/Q56E)), xvi) SEQ ID NO:72(IL-12p40(E45Q/Q56E/E59Q)), xvii) SEQ ID NO:73 (IL-12p40(D161N)), xviii)SEQ ID NO:74 (IL-12p40(E73Q)), xix) SEQ ID NO:75 (IL-12p40(Q144E)), xx)SEQ ID NO:76 (IL-12p40(E262Q)), xxi) SEQ ID NO:77 (IL-12p40(E100Q)),xxii) SEQ ID NO:78 (IL-12p40(D18N)), xxiii) SEQ ID NO:79(IL-12p40(E33Q)), xxiv) SEQ ID NO:80 (IL-12p40(Q229E)), xxv) SEQ IDNO:81 (IL-12p40(E235Q)), xxvi) SEQ ID NO:82 (IL-12p40(Q256N)), xxvii)SEQ ID NO:83 (IL-12p40(E299Q)), xxviii) SEQ ID NO:84 (IL-12p40(D87N)),xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii)IL-12p40(S43K), xxxiii) SEQ ID NO:379 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E)), xxxiv) SEQ ID NO:205(IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii)IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl)SEQ ID NO:206 (IL-12p40(E32Q/E59Q)), xli) SEQ ID NO:207(IL-12p40(D34N/E59Q)), xlii) SEQ ID NO:208 (IL-12p40(E59Q/E187Q)),xliii) SEQ ID NO:209 (IL-12p40(S43E/E59Q)), xliv) SEQ ID NO:210(IL-12p40(S43K/E49Q)), xlv) SEQ ID NO:211 (IL-12p40(E59Q/K163E)), xlvi)SEQ ID NO:212 (IL-12p40(E59Q/K99E)), xlvii) SEQ ID NO:213(IL-12p40(E59Q/K258E)), xlviii) SEQ ID NO:214 (IL-12p40(E59Q/K260E)),xlix) SEQ ID NO: 326 (IL-12p40 (D34N/E59K)), 1) SEQ ID NO: 325(IL-12p40(E59K/K99E)), li) SEQ ID NO: 339 (IL-12p40(D18K/E59K/K99E)),lii) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), liii) SEQ ID NO: 336(IL-12p40 (E59K/K99Y)), liv) SEQ ID NO: 335 (IL-12p40 (E59Y/K99E)), lv)SEQ ID NO: 338 (IL-12p40 (E45K/E59K/K99E)), lvi) SEQ ID NO: 340(IL-12p40 (E59K/K99E/Q144E)), lvii) SEQ ID NO: 341 (IL-12p40(E59K/K99E/Q144K)), lviii) SEQ ID NO: 342 (IL-12p40 (E59K/K99E/R159E)),lix) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), lx) SEQ ID NO: 344(IL-12p40 (D18K/E59K/K99E/K264E)), lxi) SEQ ID NO: 360 (IL-12p40(C252S)), lxii) SEQ ID NO: 361 (IL-12p40 (DI8K/E59K/K99E/C252S)), lxiii)SEQ ID NO: 362 (IL-12p40 (D18K/E59K/K99E/C252S/K264E)), lxiv) SEQ ID NO:363 (IL-12p40 (E59K/K99Y/C252S)), lxv) SEQ ID NO: 364 (IL-12p40(E59K/K99E/C252S/K264E)), lxvi) SEQ ID NO: 365 (IL-12p40(E59K/K99E/C252S)), lxvii) SEQ ID NO: 254 (IL-12p40 (N103D/N113D)),lxviii) SEQ ID NO: 255 (IL-12p40 (N103D/N200D)), lxix) SEQ ID NO: 256(IL-12p40 (N103D/N281D)), lxx) SEQ ID NO: 257 (IL-12p40 (N113D/N200D)),lxxi) SEQ ID NO: 258 (IL-12p40 (N113D/N281D)), lxxii) SEQ ID NO: 259(IL-12p40 (N200D/N281D)), lxxiii) SEQ ID NO: 260 (IL-12p40(N103D/N113D/N200D)), lxxiv) SEQ ID NO: 261 (IL-12p40(N103D/N113D/N281D)), lxxv) SEQ ID NO: 262 (IL-12p40(N103D/N200D/N281D)), lxxvi) SEQ ID NO: 263 (IL-12p40(N113D/N200D/N281D)), lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)),lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)), lxxix) SEQ ID NO: 266(IL-12p40 (N200Q)), lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)), lxxxi) SEQID NO: 268 (IL-12p40 (N103Q/N113Q)), lxxxii) SEQ ID NO: 269 (IL-12p40(N103Q/N200Q)), lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q/N281Q)),lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q/N200Q)), lxxxv) SEQ ID NO: 272(IL-12p40 (N113Q/N281Q)), lxxxvi) SEQ ID NO: 273 (IL-12p40(N200Q/N281Q)), lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q/N113Q/N200Q)),lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q/N113Q/N281Q)), lxxxix) SEQ IDNO: 276 (IL-12p40 (N103Q/N200Q/N281Q)), xc) SEQ ID NO: 277 (IL-12p40(N113Q/N200Q/N281Q)), xci) SEQ ID NO:278 (IL-12p40(N103Q/N113Q/N200Q/N281Q)), xcii) SEQ ID NO:327 (IL-12p40(D34N/E59K/K99E)), xciii) SEQ ID NO:328 (IL-12p40 (D34K/E59K/K99E)),xciv) SEQ ID NO:329 (IL-12p40 (E32Q/D34N/E59K/K99E)), xcv) SEQ ID NO:331(IL-12p40 (E32K/D34N/E59K/K99E)), xcvi) SEQ ID NO: 337 (IL-12p40(E59Y/K99Y)), xcvii) SEQ ID NO:366 (IL-12p40(E59K/K99E/N103Q/C252S/K264E)), xcviii) SEQ ID NO:367 (IL-12p40(E59K/K99E/N113Q/C252S/K264E)), xcix) SEQ ID NO:368 (IL-12p40(E59K/K99E/N200Q/C252S/K264E)), c) SEQ ID NO:369 (IL-12p40(E59K/K99E/N281Q/C252S/K264E)), ci) SEQ ID NO:370 (IL-12p40(E59K/K99E/N103Q/N113Q/C252S/K264E)), cii) SEQ ID NO:371 (IL-12p40(E59K/K99E/N103Q/N200Q/C252S/K264E)), ciii) SEQ ID NO:372 (IL-12p40(E59K/K99E/N103Q/N281Q/C252S/K264E)), civ) SEQ ID NO:373 (IL-12p40(E59K/K99E/N113Q/N200Q/C252S/K264E)), cv) SEQ ID NO:374 (IL-12p40(E59K/K99E/N113Q/N281Q/C252S/K264E)), cvi) SEQ ID NO:375 (IL-12p40(E59K/K99E/N200Q/N281Q/C252S/K264E)), cvii) SEQ ID NO:376 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E)), cviii) SEQ ID NO:377(IL-12p40 (E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E)), and cix) SEQ IDNO:378 (IL-12p40 (E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E)).

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidmodifications as amino acid residues selected from the group consistingof Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63,L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97,L124, M125, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163,D165, I171, R181, 1182, R183, V185, T186, D188, R189, V190, S192, Y193,N195, and A196.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidsubstitutions selected from the group consisting of N21D, Q35D, E38Q,D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A,L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K,E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p35 subunit has amino acidsubstitutions selected from the group consisting of N71D/N85D/N195D,N151D/E153Q, N151D/D165N, Q130E/N151D, N151D/K158E, E79Q/N151D,D55Q/N151D, N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q, N71Q/N195Q,N85Q/N195Q, N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, and N85D/N195D.

In some embodiments, the present invention provides a heterodimeric Fcfusion protein wherein said IL-12p35 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:113(IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115(IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D/N85D/N195D)), v) SEQID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii)SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(Q135E)),ix) SEQ ID NO:121 (IL-12p35(Q35D)), x) SEQ ID NO:122 (IL-12p35(Q146E)),xi) SEQ ID NO:123 (IL-12p35(N76D)), xii) SEQ ID NO:124(IL-12p35(E162Q)), xiii) SEQ ID NO:125 (IL-12p35(E163Q)), xiv)IL-12p35(N21D), xv) SEQ ID NO:333 (IL-12p35(D55Q)), xvi) IL-12p35(E79Q),xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx)SEQ ID NO:227 (IL-12p35(N151K)), xxi) SEQ ID NO:226 (IL-12p35(E153K)),xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO:225(IL-12p35(N151D/E153Q)), xxv) SEQ ID NO:228 (IL-12p35(N151D/D165N)),xxvi) SEQ ID NO:229 (IL-12p35(Q130E/N151D)), xxvii) SEQ ID NO:230(IL-12p35(N151D/K158E)), xxviii) SEQ ID NO:231 (IL-12p35(E79Q/N151D)),xxix) SEQ ID NO:232 (IL-12p35(D55Q/N151D)), xxx) SEQ ID NO:233(IL-12p35(N136D/N151D)), xxxi) SEQ ID NO:234 (IL-12p35(N21D/N151D)),xxxii) SEQ ID NO:235 (IL-12p35(E143Q/N151D)), xxxiii) SEQ ID NO: 345(IL-12p35(F96A)), xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)), xxxv) SEQ IDNO: 347 (IL-12p35(L89A)), xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)),xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)), xxxviii) SEQ ID NO: 350(IL-12p35(L75A)), xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)), xxxv) SEQ IDNO: 279 (IL-12p35 (N71Q)), xxxvi) SEQ ID NO: 280 (IL-12p35 (N85Q)),xxxvii) SEQ ID NO: 281 (IL-12p35 (N195Q)), xxxviii) SEQ ID NO: 282(IL-12p35 (N71Q/N85Q)), xxxix) SEQ ID NO: 283 (IL-12p35 (N71Q/N195Q)),xl) SEQ ID NO: 284 (IL-12p35 (N85Q/N195Q), xli) SEQ ID NO: 285 (IL-12p35(N71Q/N85Q/N195Q)), xlii) SEQ ID NO: 286 (IL-12p35 (N71D/N85D)), xliii)SEQ ID NO: 287 (IL-12p35 (N71D/N195D), xliv) SEQ ID NO: 288 (IL-12p35(N85D/N195D)), xlv) SEQ ID NO: 333 (IL-12p35 (D55Q)), and xlvi) SEQ IDNO: 334 (IL-12p35 (D55K)).

In some embodiments, the present invention provides a compositioncomprising a heterodimeric Fc fusion protein for use in treating cancerin a subject.

In some embodiments, the present invention provides one or more nucleicacids encoding a heterodimeric Fc fusion protein.

In some embodiments, the present invention provides a host cellcomprising said one or more nucleic acids encoding a heterodimeric Fcfusion protein.

In some embodiments, the present invention provides a method of making aheterodimeric Fc fusion protein, said method comprising culturing a hostcell under conditions whereby said heterodimeric Fc fusion protein isproduced.

In some embodiments, the present invention provides a method ofpurifying a heterodimeric Fc fusion protein, said method comprising: a)providing a composition comprising the heterodimeric Fc fusion protein;b) loading said composition onto an ion exchange column; and c)collecting a fraction containing said heterodimeric Fc fusion protein.

In another aspect, the present invention provides a heterodimericcomplex comprising: a) an IL-12p40 subunit, and b) an IL-12p35 subunit.

In some embodiments, the present invention provides a heterodimericcomplex wherein said IL-12p40 subunit is covalently attached to saidIL-12p35 subunit using domain linker.

In some embodiments, the present invention provides a heterodimericcomplex wherein said IL-12p40 subunit has a polypeptide sequenceselected form the group consisting of SEQ ID NO:3 (human IL-12 subunitbeta (IL-12p40) precursor sequence) and SEQ ID NO:4 (human IL-12 subunitbeta (IL-12p40) mature form sequence), and/or said IL-12p35 subunit hasa polypeptide sequence selected from the group consisting of SEQ ID NO:1(human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ IDNO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

In some embodiments, the present invention provides a heterodimericcomplex wherein said first and second Fc domains further comprise aminoacid substitutions M428L/N434S.

In some embodiments, the present invention provides a heterodimericcomplex wherein said IL-12p40 subunit is a variant IL-12p40 subunitand/or said IL-12p35 subunit is a variant IL-12p35 subunit.

In some embodiments, the present invention provides a heterodimericcomplex wherein said IL-12p40 subunit is a variant IL-12p40 subunithaving altered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1),IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12 receptor complex;and/or said IL-12p35 subunit is a variant IL-12p35 subunit havingaltered affinity for IL-12 receptor subunit beta-1 (IL-12Rβ1), IL-12receptor subunit beta-2 (IL-12Rβ2), and/or IL-12 receptor complex.

In some embodiments, the present invention provides a heterodimericcomplex wherein said IL-12p40 subunit has one or more amino acidmodifications at amino acid residues selected from the group consistingof E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32,E33, D34, L40, D41, Q42, S43, E45, L47, T54, 155, Q56, K58, E59, F60,G61, D62, Q65, Y66, E73, K84, E86, D87, G88, 189, W90, D93, D97, K99,E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156,R159, D161, N162, K163, D166, D170, Q172, D174, A176, C177, P178, A179,A180, E181, S183, P185, E187, N200, S204, F206, R208, D209, D214, N218,Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F247, S248, C252,Q256, K258, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292,Y293, and E299.

In some embodiments, the present invention provides a heterodimericcomplex wherein said IL-12p40 subunit has one or more amino acidsubstitutions selected from the group consisting of D18N, D18K, E32Q,E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q,D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N,R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N,K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

In some embodiments, the present invention provides a heterodimericcomplex wherein said IL-12p40 subunit has amino acid substitutionsselected from the group consisting of N103D/N113D/N200D/N281D,Q42E/E45Q, E45Q/Q56E, Q42E/E59Q, Q56E/E59Q, Q42E/E45Q/Q56E,E45Q/Q56E/E59Q, E32Q/E59Q, D34N/E59K, D34N/E59K/K99E, D34K/E59K/K99E,E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q, E59Q/E187Q,S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E, E59Q/K260E,E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y, E59Y/K99Y,E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In some embodiments, the present invention provides a heterodimericcomplex wherein said IL-12p40 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:57(IL-12p40(N103D)), ii) SEQ ID NO:58 (IL-12p40(N113D)), iii) SEQ ID NO:59(IL-12p40(N200D)), iv) SEQ ID NO:60 (IL-12p40(N281D)), v) SEQ ID NO:61(IL-12p40(N103D/N113D/N200D/N281D)), vi) SEQ ID NO:62 (IL-12p40(Q42E)),vii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)),ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi)SEQ ID NO:67 (IL-12p40(Q42E/E45Q)), xii) SEQ ID NO:68(IL-12p40(E45Q/Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E/E59Q)), xiv)SEQ ID NO:70 (IL-12p40(Q56E/E59Q)), xv) SEQ ID NO:71(IL-12p40(Q42E/E45Q/Q56E)), xvi) SEQ ID NO:72(IL-12p40(E45Q/Q56E/E59Q)), xvii) SEQ ID NO:73 (IL-12p40(D161N)), xviii)SEQ ID NO:74 (IL-12p40(E73Q)), xix) SEQ ID NO:75 (IL-12p40(Q144E)), xx)SEQ ID NO:76 (IL-12p40(E262Q)), xxi) SEQ ID NO:77 (IL-12p40(E100Q)),xxii) SEQ ID NO:78 (IL-12p40(D18N)), xxiii) SEQ ID NO:79(IL-12p40(E33Q)), xxiv) SEQ ID NO:80 (IL-12p40(Q229E)), xxv) SEQ IDNO:81 (IL-12p40(E235Q)), xxvi) SEQ ID NO:82 (IL-12p40(Q256N)), xxvii)SEQ ID NO:83 (IL-12p40(E299Q)), xxviii) SEQ ID NO:84 (IL-12p40(D87N)),xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii)IL-12p40(S43K), xxxiii) SEQ ID NO:379 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E)), xxxiv) SEQ ID NO:205(IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii)IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl)SEQ ID NO:206 (IL-12p40(E32Q/E59Q)), xli) SEQ ID NO:207(IL-12p40(D34N/E59Q)), xlii) SEQ ID NO:208 (IL-12p40(E59Q/E187Q)),xliii) SEQ ID NO:209 (IL-12p40(S43E/E59Q)), xliv) SEQ ID NO:210(IL-12p40(S43K/E49Q)), xlv) SEQ ID NO:211 (IL-12p40(E59Q/K163E)), xlvi)SEQ ID NO:212 (IL-12p40(E59Q/K99E)), xlvii) SEQ ID NO:213(IL-12p40(E59Q/K258E)), xlviii) SEQ ID NO:214 (IL-12p40(E59Q/K260E)),xlix) SEQ ID NO: 326 (IL-12p40 (D34N/E59K)), 1) SEQ ID NO: 325(IL-12p40(E59K/K99E)), li) SEQ ID NO: 339 (IL-12p40(D18K/E59K/K99E)),lii) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), liii) SEQ ID NO: 336(IL-12p40 (E59K/K99Y)), liv) SEQ ID NO: 335 (IL-12p40 (E59Y/K99E)), lv)SEQ ID NO: 338 (IL-12p40 (E45K/E59K/K99E)), lvi) SEQ ID NO: 340(IL-12p40 (E59K/K99E/Q144E)), lvii) SEQ ID NO: 341 (IL-12p40(E59K/K99E/Q144K)), lviii) SEQ ID NO: 342 (IL-12p40 (E59K/K99E/R159E)),lix) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), lx) SEQ ID NO: 344(IL-12p40 (D18K/E59K/K99E/K264E)), lxi) SEQ ID NO: 360 (IL-12p40(C252S)), lxii) SEQ ID NO: 361 (IL-12p40 (DI8K/E59K/K99E/C252S)), lxiii)SEQ ID NO: 362 (IL-12p40 (D18K/E59K/K99E/C252S/K264E)), lxiv) SEQ ID NO:363 (IL-12p40 (E59K/K99Y/C252S)), lxv) SEQ ID NO: 364 (IL-12p40(E59K/K99E/C252S/K264E)), lxvi) SEQ ID NO: 365 (IL-12p40(E59K/K99E/C252S)), lxvii) SEQ ID NO: 254 (IL-12p40 (N103D/N113D)),lxviii) SEQ ID NO: 255 (IL-12p40 (N103D/N200D)), lxix) SEQ ID NO: 256(IL-12p40 (N103D/N281D)), lxx) SEQ ID NO: 257 (IL-12p40 (N113D/N200D)),lxxi) SEQ ID NO: 258 (IL-12p40 (N113D/N281D)), lxxii) SEQ ID NO: 259(IL-12p40 (N200D/N281D)), lxxiii) SEQ ID NO: 260 (IL-12p40(N103D/N113D/N200D)), lxxiv) SEQ ID NO: 261 (IL-12p40(N103D/N113D/N281D)), lxxv) SEQ ID NO: 262 (IL-12p40(N103D/N200D/N281D)), lxxvi) SEQ ID NO: 263 (IL-12p40(N113D/N200D/N281D)), lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)),lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)), lxxix) SEQ ID NO: 266(IL-12p40 (N200Q)), lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)), lxxxi) SEQID NO: 268 (IL-12p40 (N103Q/N113Q)), lxxxii) SEQ ID NO: 269 (IL-12p40(N103Q/N200Q)), lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q/N281Q)),lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q/N200Q)), lxxxv) SEQ ID NO: 272(IL-12p40 (N113Q/N281Q)), lxxxvi) SEQ ID NO: 273 (IL-12p40(N200Q/N281Q)), lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q/N113Q/N200Q)),lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q/N113Q/N281Q)), lxxxix) SEQ IDNO: 276 (IL-12p40 (N103Q/N200Q/N281Q)), xc) SEQ ID NO: 277 (IL-12p40(N113Q/N200Q/N281Q)), xci) SEQ ID NO:278 (IL-12p40(N103Q/N113Q/N200Q/N281Q)), xcii) SEQ ID NO:327 (IL-12p40(D34N/E59K/K99E)), xciii) SEQ ID NO:328 (IL-12p40 (D34K/E59K/K99E)),xciv) SEQ ID NO:329 (IL-12p40 (E32Q/D34N/E59K/K99E)), xcv) SEQ ID NO:331(IL-12p40 (E32K/D34N/E59K/K99E)), xcvi) SEQ ID NO: 337 (IL-12p40(E59Y/K99Y)), xcvii) SEQ ID NO:366 (IL-12p40(E59K/K99E/N103Q/C252S/K264E)), xcviii) SEQ ID NO:367 (IL-12p40(E59K/K99E/N113Q/C252S/K264E)), xcix) SEQ ID NO:368 (IL-12p40(E59K/K99E/N200Q/C252S/K264E)), c) SEQ ID NO:369 (IL-12p40(E59K/K99E/N281Q/C252S/K264E)), ci) SEQ ID NO:370 (IL-12p40(E59K/K99E/N103Q/N113Q/C252S/K264E)), cii) SEQ ID NO:371 (IL-12p40(E59K/K99E/N103Q/N200Q/C252S/K264E)), ciii) SEQ ID NO:372 (IL-12p40(E59K/K99E/N103Q/N281Q/C252S/K264E)), civ) SEQ ID NO:373 (IL-12p40(E59K/K99E/N113Q/N200Q/C252S/K264E)), cv) SEQ ID NO:374 (IL-12p40(E59K/K99E/N113Q/N281Q/C252S/K264E)), cvi) SEQ ID NO:375 (IL-12p40(E59K/K99E/N200Q/N281Q/C252S/K264E)), cvii) SEQ ID NO:376 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E)), cviii) SEQ ID NO:377(IL-12p40 (E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E)), and cix) SEQ IDNO:378 (IL-12p40 (E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E)).

In some embodiments, the present invention provides a heterodimericcomplex wherein said IL-12p35 subunit has one or more amino acidmodifications as amino acid residues selected from the group consistingof Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63,L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97,L124, M125, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163,D165, I171, R181, 1182, R183, V185, T186, D188, R189, V190, S192, Y193,N195, and A196.

In some embodiments, the present invention provides a heterodimericcomplex wherein said IL-12p35 subunit has one or more amino acidsubstitutions selected from the group consisting of N21D, Q35D, E38Q,D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A,L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K,E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

In some embodiments, the present invention provides a heterodimericcomplex wherein said IL-12p35 subunit has amino acid substitutionsselected from the group consisting of N71D/N85D/N195D, N151D/E153Q,N151D/D165N, Q130E/N151D, N151D/K158E, E79Q/N151D, D55Q/N151D,N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q, N71Q/N195Q, N85Q/N195Q,N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, and N85D/N195D.

In some embodiments, the present invention provides a heterodimericcomplex wherein said IL-12p35 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:113(IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115(IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D/N85D/N195D)), v) SEQID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii)SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(Q135E)),ix) SEQ ID NO:121 (IL-12p35(Q35D)), x) SEQ ID NO:122 (IL-12p35(Q146E)),xi) SEQ ID NO:123 (IL-12p35(N76D)), xii) SEQ ID NO:124(IL-12p35(E162Q)), xiii) SEQ ID NO:125 (IL-12p35(E163Q)), xiv)IL-12p35(N21D), xv) SEQ ID NO:333 (IL-12p35(D55Q)), xvi) IL-12p35(E79Q),xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx)SEQ ID NO:227 (IL-12p35(N151K)), xxi) SEQ ID NO:226 (IL-12p35(E153K)),xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO:225(IL-12p35(N151D/E153Q)), xxv) SEQ ID NO:228 (IL-12p35(N151D/D165N)),xxvi) SEQ ID NO:229 (IL-12p35(Q130E/N151D)), xxvii) SEQ ID NO:230(IL-12p35(N151D/K158E)), xxviii) SEQ ID NO:231 (IL-12p35(E79Q/N151D)),xxix) SEQ ID NO:232 (IL-12p35(D55Q/N151D)), xxx) SEQ ID NO:233(IL-12p35(N136D/N151D)), xxxi) SEQ ID NO:234 (IL-12p35(N21D/N151D)),xxxii) SEQ ID NO:235 (IL-12p35(E143Q/N151D)), xxxiii) SEQ ID NO: 345(IL-12p35(F96A)), xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)), xxxv) SEQ IDNO: 347 (IL-12p35(L89A)), xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)),xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)), xxxviii) SEQ ID NO: 350(IL-12p35(L75A)), xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)), xxxv) SEQ IDNO: 279 (IL-12p35 (N71Q)), xxxvi) SEQ ID NO: 280 (IL-12p35 (N85Q)),xxxvii) SEQ ID NO: 281 (IL-12p35 (N195Q)), xxxviii) SEQ ID NO: 282(IL-12p35 (N71Q/N85Q)), xxxix) SEQ ID NO: 283 (IL-12p35 (N71Q/N195Q)),xl) SEQ ID NO: 284 (IL-12p35 (N85Q/N195Q), xli) SEQ ID NO: 285 (IL-12p35(N71Q/N85Q/N195Q)), xlii) SEQ ID NO: 286 (IL-12p35 (N71D/N85D)), xliii)SEQ ID NO: 287 (IL-12p35 (N71D/N195D), xliv) SEQ ID NO: 288 (IL-12p35(N85D/N195D)), xlv) SEQ ID NO: 333 (IL-12p35 (D55Q)), and xlvi) SEQ IDNO: 334 (IL-12p35 (D55K)).

In some embodiments, the present invention provides a compositioncomprising a heterodimeric complex for use in treating cancer in asubject.

In some embodiments, the present invention provides one or more nucleicacids encoding a heterodimeric complex.

In some embodiments, the present invention provides a host cellcomprising said one or more nucleic acids encoding a heterodimericcomplex.

In some embodiments, the present invention provides a method of making aheterodimeric complex, said method comprising culturing a host cellunder conditions whereby said heterodimeric complex is produced.

In some embodiments, the present invention provides a method ofpurifying a heterodimeric complex, said method comprising: a) providinga composition comprising the heterodimeric complex; b) loading saidcomposition onto an ion exchange column; and c) collecting a fractioncontaining said heterodimeric complex.

In another aspect, the present invention provides a variant IL-12p40subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex.

In some embodiments, the present invention provides a variant IL-12p40subunit wherein said variant IL-12p40 subunit has one or more amino acidmodifications at amino acid residues selected from the group consistingof E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32,E33, D34, L40, D41, Q42, S43, E45, L47, T54, 155, Q56, K58, E59, F60,G61, D62, Q65, Y66, E73, K84, E86, D87, G88, 189, W90, D93, D97, K99,E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156,R159, D161, N162, K163, D166, D170, Q172, D174, A176, C177, P178, A179,A180, E181, S183, P185, E187, N200, S204, F206, R208, D209, D214, N218,Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F247, S248, Q256,C252, K258, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292,Y293, and E299.

In some embodiments, the present invention provides a variant IL-12p40subunit wherein said variant IL-12p40 subunit has one or more amino acidsubstitutions selected from the group consisting of D18N, D18K, E32Q,E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q,D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N,R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N,K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

In some embodiments, the present invention provides a variant IL-12p40subunit wherein said variant IL-12p40 subunit has amino acidsubstitutions selected from the group consisting ofN103D/N113D/N200D/N281D, Q42E/E45Q, E45Q/Q56E, Q42E/E59Q, Q56E/E59Q,Q42E/E45Q/Q56E, E45Q/Q56E/E59Q, E32Q/E59Q, D34N/E59K, D34N/E59K/K99E,D34K/E59K/K99E, E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q,E59Q/E187Q, S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E,E59Q/K260E, E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y,E59Y/K99Y, E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In some embodiments, the present invention provides a variant IL-12p40subunit wherein said variant IL-12p40 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:57(IL-12p40(N103D)), ii) SEQ ID NO:58 (IL-12p40(N113D)), iii) SEQ ID NO:59(IL-12p40(N200D)), iv) SEQ ID NO:60 (IL-12p40(N281D)), v) SEQ ID NO:61(IL-12p40(N103D/N113D/N200D/N281D)), vi) SEQ ID NO:62 (IL-12p40(Q42E)),vii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)),ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi)SEQ ID NO:67 (IL-12p40(Q42E/E45Q)), xii) SEQ ID NO:68(IL-12p40(E45Q/Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E/E59Q)), xiv)SEQ ID NO:70 (IL-12p40(Q56E/E59Q)), xv) SEQ ID NO:71(IL-12p40(Q42E/E45Q/Q56E)), xvi) SEQ ID NO:72(IL-12p40(E45Q/Q56E/E59Q)), xvii) SEQ ID NO:73 (IL-12p40(D161N)), xviii)SEQ ID NO:74 (IL-12p40(E73Q)), xix) SEQ ID NO:75 (IL-12p40(Q144E)), xx)SEQ ID NO:76 (IL-12p40(E262Q)), xxi) SEQ ID NO:77 (IL-12p40(E100Q)),xxii) SEQ ID NO:78 (IL-12p40(D18N)), xxiii) SEQ ID NO:79(IL-12p40(E33Q)), xxiv) SEQ ID NO:80 (IL-12p40(Q229E)), xxv) SEQ IDNO:81 (IL-12p40(E235Q)), xxvi) SEQ ID NO:82 (IL-12p40(Q256N)), xxvii)SEQ ID NO:83 (IL-12p40(E299Q)), xxviii) SEQ ID NO:84 (IL-12p40(D87N)),xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii)IL-12p40(S43K), xxxiii) SEQ ID NO:379 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E)), xxxiv) SEQ ID NO:205(IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii)IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl)SEQ ID NO:206 (IL-12p40(E32Q/E59Q)), xli) SEQ ID NO:207(IL-12p40(D34N/E59Q)), xlii) SEQ ID NO:208 (IL-12p40(E59Q/E187Q)),xliii) SEQ ID NO:209 (IL-12p40(S43E/E59Q)), xliv) SEQ ID NO:210(IL-12p40(S43K/E49Q)), xlv) SEQ ID NO:211 (IL-12p40(E59Q/K163E)), xlvi)SEQ ID NO:212 (IL-12p40(E59Q/K99E)), xlvii) SEQ ID NO:213(IL-12p40(E59Q/K258E)), xlviii) SEQ ID NO:214 (IL-12p40(E59Q/K260E)),xlix) SEQ ID NO: 326 (IL-12p40 (D34N/E59K)), 1) SEQ ID NO: 325(IL-12p40(E59K/K99E)), li) SEQ ID NO: 339 (IL-12p40(D18K/E59K/K99E)),lii) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), liii) SEQ ID NO: 336(IL-12p40 (E59K/K99Y)), liv) SEQ ID NO: 335 (IL-12p40 (E59Y/K99E)), lv)SEQ ID NO: 338 (IL-12p40 (E45K/E59K/K99E)), lvi) SEQ ID NO: 340(IL-12p40 (E59K/K99E/Q144E)), lvii) SEQ ID NO: 341 (IL-12p40(E59K/K99E/Q144K)), lviii) SEQ ID NO: 342 (IL-12p40 (E59K/K99E/R159E)),lix) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), lx) SEQ ID NO: 344(IL-12p40 (D18K/E59K/K99E/K264E)), lxi) SEQ ID NO: 360 (IL-12p40(C252S)), lxii) SEQ ID NO: 361 (IL-12p40 (DI8K/E59K/K99E/C252S)), lxiii)SEQ ID NO: 362 (IL-12p40 (D18K/E59K/K99E/C252S/K264E)), lxiv) SEQ ID NO:363 (IL-12p40 (E59K/K99Y/C252S)), lxv) SEQ ID NO: 364 (IL-12p40(E59K/K99E/C252S/K264E)), lxvi) SEQ ID NO: 365 (IL-12p40(E59K/K99E/C252S)), lxvii) SEQ ID NO: 254 (IL-12p40 (N103D/N113D)),lxviii) SEQ ID NO: 255 (IL-12p40 (N103D/N200D)), lxix) SEQ ID NO: 256(IL-12p40 (N103D/N281D)), lxx) SEQ ID NO: 257 (IL-12p40 (N113D/N200D)),lxxi) SEQ ID NO: 258 (IL-12p40 (N113D/N281D)), lxxii) SEQ ID NO: 259(IL-12p40 (N200D/N281D)), lxxiii) SEQ ID NO: 260 (IL-12p40(N103D/N113D/N200D)), lxxiv) SEQ ID NO: 261 (IL-12p40(N103D/N113D/N281D)), lxxv) SEQ ID NO: 262 (IL-12p40(N103D/N200D/N281D)), lxxvi) SEQ ID NO: 263 (IL-12p40(N113D/N200D/N281D)), lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)),lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)), lxxix) SEQ ID NO: 266(IL-12p40 (N200Q)), lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)), lxxxi) SEQID NO: 268 (IL-12p40 (N103Q/N113Q)), lxxxii) SEQ ID NO: 269 (IL-12p40(N103Q/N200Q)), lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q/N281Q)),lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q/N200Q)), lxxxv) SEQ ID NO: 272(IL-12p40 (N113Q/N281Q)), lxxxvi) SEQ ID NO: 273 (IL-12p40(N200Q/N281Q)), lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q/N113Q/N200Q)),lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q/N113Q/N281Q)), lxxxix) SEQ IDNO: 276 (IL-12p40 (N103Q/N200Q/N281Q)), xc) SEQ ID NO: 277 (IL-12p40(N113Q/N200Q/N281Q)), xci) SEQ ID NO:278 (IL-12p40(N103Q/N113Q/N200Q/N281Q)), xcii) SEQ ID NO:327 (IL-12p40(D34N/E59K/K99E)), xciii) SEQ ID NO:328 (IL-12p40 (D34K/E59K/K99E)),xciv) SEQ ID NO:329 (IL-12p40 (E32Q/D34N/E59K/K99E)), xcv) SEQ ID NO:331(IL-12p40 (E32K/D34N/E59K/K99E)), xcvi) SEQ ID NO: 337 (IL-12p40(E59Y/K99Y)), xcvii) SEQ ID NO:366 (IL-12p40(E59K/K99E/N103Q/C252S/K264E)), xcviii) SEQ ID NO:367 (IL-12p40(E59K/K99E/N113Q/C252S/K264E)), xcix) SEQ ID NO:368 (IL-12p40(E59K/K99E/N200Q/C252S/K264E)), c) SEQ ID NO:369 (IL-12p40(E59K/K99E/N281Q/C252S/K264E)), ci) SEQ ID NO:370 (IL-12p40(E59K/K99E/N103Q/N113Q/C252S/K264E)), cii) SEQ ID NO:371 (IL-12p40(E59K/K99E/N103Q/N200Q/C252S/K264E)), ciii) SEQ ID NO:372 (IL-12p40(E59K/K99E/N103Q/N281Q/C252S/K264E)), civ) SEQ ID NO:373 (IL-12p40(E59K/K99E/N113Q/N200Q/C252S/K264E)), cv) SEQ ID NO:374 (IL-12p40(E59K/K99E/N113Q/N281Q/C252S/K264E)), cvi) SEQ ID NO:375 (IL-12p40(E59K/K99E/N200Q/N281Q/C252S/K264E)), cvii) SEQ ID NO:376 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E)), cviii) SEQ ID NO:377(IL-12p40 (E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E)), and cix) SEQ IDNO:378 (IL-12p40 (E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E)).

In some embodiments, the present invention provides a compositioncomprising a variant IL-12p40 subunit.

In some embodiments, the present invention provides a nucleic acidencoding a variant IL-12p40 subunit.

In some embodiments, the present invention provides a host cellcomprising said nucleic acid encoding a variant IL-12p40 subunit.

In some embodiments, the present invention provides a method of making avariant IL-12p40 subunit, said method comprising culturing a host cellaccording to claim H8 under conditions whereby said variant IL-12p40subunit is produced.

In another aspect, the present invention provides a variant IL-12p35subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ32), and/or IL-12receptor complex.

In some embodiments, the present invention provides a variant IL-12p35subunit, wherein said variant IL-12p35 subunit has one or more aminoacid modifications as amino acid residues selected from the groupconsisting of Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59,V60, E61, C63, L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85,L89, F96, M97, L124, M125, Q130, Q135, N136, E143, Q146, N151, E153,K158, E162, E163, D165, I171, R181, 1182, R183, V185, T186, D188, R189,V190, S192, Y193, N195, and A196.

In some embodiments, the present invention provides a variant IL-12p35subunit, wherein said variant IL-12p35 subunit has one or more aminoacid substitutions selected from the group consisting of N21D, Q35D,E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A,M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K,E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

In some embodiments, the present invention provides a variant IL-12p35subunit, wherein said variant IL-12p35 subunit amino acid substitutionsselected from the group consisting of N71D/N85D/N195D, N151D/E153Q,N151D/D165N, Q130E/N151D, N151D/K158E, E79Q/N151D, D55Q/N151D,N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q, N71Q/N195Q, N85Q/N195Q,N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, and N85D/N195D.

In some embodiments, the present invention provides a variant IL-12p35subunit, wherein said variant IL-12p35 subunit has amino acidsubstitutions N71D/N85D/N195D.

In some embodiments, the present invention provides a variant IL-12p35subunit wherein said variant IL-12p35 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:113(IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115(IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D/N85D/N195D)), v) SEQID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii)SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(Q135E)),ix) SEQ ID NO:121 (IL-12p35(Q35D)), x) SEQ ID NO:122 (IL-12p35(Q146E)),xi) SEQ ID NO:123 (IL-12p35(N76D)), xii) SEQ ID NO:124(IL-12p35(E162Q)), xiii) SEQ ID NO:125 (IL-12p35(E163Q)), xiv)IL-12p35(N21D), xv) SEQ ID NO:333 (IL-12p35(D55Q)), xvi) IL-12p35(E79Q),xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx)SEQ ID NO:227 (IL-12p35(N151K)), xxi) SEQ ID NO:226 (IL-12p35(E153K)),xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO:225(IL-12p35(N151D/E153Q)), xxv) SEQ ID NO:228 (IL-12p35(N151D/D165N)),xxvi) SEQ ID NO:229 (IL-12p35(Q130E/N151D)), xxvii) SEQ ID NO:230(IL-12p35(N151D/K158E)), xxviii) SEQ ID NO:231 (IL-12p35(E79Q/N151D)),xxix) SEQ ID NO:232 (IL-12p35(D55Q/N151D)), xxx) SEQ ID NO:233(IL-12p35(N136D/N151D)), xxxi) SEQ ID NO:234 (IL-12p35(N21D/N151D)),xxxii) SEQ ID NO:235 (IL-12p35(E143Q/N151D)), xxxiii) SEQ ID NO: 345(IL-12p35(F96A)), xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)), xxxv) SEQ IDNO: 347 (IL-12p35(L89A)), xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)),xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)), xxxviii) SEQ ID NO: 350(IL-12p35(L75A)), xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)), xxxv) SEQ IDNO: 279 (IL-12p35 (N71Q)), xxxvi) SEQ ID NO: 280 (IL-12p35 (N85Q)),xxxvii) SEQ ID NO: 281 (IL-12p35 (N195Q)), xxxviii) SEQ ID NO: 282(IL-12p35 (N71Q/N85Q)), xxxix) SEQ ID NO: 283 (IL-12p35 (N71Q/N195Q)),xl) SEQ ID NO: 284 (IL-12p35 (N85Q/N195Q), xli) SEQ ID NO: 285 (IL-12p35(N71Q/N85Q/N195Q)), xlii) SEQ ID NO: 286 (IL-12p35 (N71D/N85D)), xliii)SEQ ID NO: 287 (IL-12p35 (N71D/N195D), xliv) SEQ ID NO: 288 (IL-12p35(N85D/N195D)), xlv) SEQ ID NO: 333 (IL-12p35 (D55Q)), and xlvi) SEQ IDNO: 334 (IL-12p35 (D55K)).

In some embodiments, the present invention provides a compositioncomprising a variant IL-12p35 subunit.

In some embodiments, the present invention provides a nucleic acidencoding a variant IL-12p35 subunit.

In some embodiments, the present invention provides a host cellcomprising said nucleic acid encoding a variant IL-12p35 subunit.

In some embodiments, the present invention provides a variant IL-12p35subunit, said method comprising culturing a host cell under conditionswhereby said variant IL-12p35 subunit is produced.

In another aspect, the present invention provides a heterodimericprotein comprising: a) a first fusion protein comprising an IL-12p40subunit domain covalently attached to a first Fc domain; and b) a secondfusion protein comprising an IL-12p35 subunit domain covalent attachedto a second Fc domain; wherein said first and second Fc domains a set ofamino acid substitutions selected from the group consisting ofL368D/K370S and S364K; L368D/K370S and S364K/E357L; L368D/K370S andS364K/E357Q; T411E/K360E/Q362E and D401K; L368E/K370S and S364K; K370Sand S364K/E357Q; T366S/L368A/Y407V and T366W; andT366S/L368A/Y407V/Y349C and T366W/S354C; according to EU numbering.

In some embodiments, the present invention provides a heterodimericprotein, wherein said IL-12p40 subunit domain is attached N-terminal tosaid first Fc domain and said IL-12p35 subunit domain is attachedN-terminal to said second Fc domain.

In some embodiments, the present invention provides a heterodimericprotein, wherein said IL-12p40 subunit domain is attached C-terminal tosaid first Fc domain and said IL-12p35 subunit domain is attachedC-terminal to said second Fc domain.

In some embodiments, the present invention provides a heterodimericprotein, wherein said Il-12p40 subunit domain is attached to said firstFc domain using a domain linker and said IL-12p35 subunit domain isattached to said second Fc domain using a domain linker.

In some embodiments, the present invention provides a heterodimericprotein wherein said first and second Fc domain comprises the IgG1hinge-CH2-CH3.

In some embodiments, the present invention provides a heterodimericprotein, wherein said first fusion protein comprises, from N- toC-terminal, an IL-12p40 subunit domain-domain linker-hinge-CH2-CH3 andsaid second fusion protein comprises, from N- to C-terminal, an IL-12p35subunit domain-domain linker-hinge-CH2-CH3.

In some embodiments, the present invention provides a heterodimericprotein, wherein said first fusion protein comprises, from N- toC-terminal, hinge-CH2-CH3-domain linker-IL-12p40 subunit domain and saidsecond fusion protein comprises, from N- to C-terminal,hinge-CH2-CH3-domain linker-IL-12p35 subunit domain.

In some embodiments, the present invention provides a heterodimericprotein wherein said IL-12p40 subunit has a sequence selected from thegroup consisting of: i) SEQ ID NO:57 (IL-12p40(N103D)), ii) SEQ ID NO:58(IL-12p40(N113D)), iii) SEQ ID NO:59 (IL-12p40(N200D)), iv) SEQ ID NO:60(IL-12p40(N281D)), v) SEQ ID NO:61 (IL-12p40(N103D/N113D/N200D/N281D)),vi) SEQ ID NO:62 (IL-12p40(Q42E)), vii) SEQ ID NO:63 (IL-12p40(E45Q)),viii) SEQ ID NO:64 (IL-12p40(Q56E)), ix) SEQ ID NO:65 (IL-12p40(E59Q)),x) SEQ ID NO:66 (IL-12p40(D62N)), xi) SEQ ID NO:67(IL-12p40(Q42E/E45Q)), xii) SEQ ID NO:68 (IL-12p40(E45Q/Q56E)), xiii)SEQ ID NO:69 (IL-12p40(Q42E/E59Q)), xiv) SEQ ID NO:70(IL-12p40(Q56E/E59Q)), xv) SEQ ID NO:71 (IL-12p40(Q42E/E45Q/Q56E)), xvi)SEQ ID NO:72 (IL-12p40(E45Q/Q56E/E59Q)), xvii) SEQ ID NO:73(IL-12p40(D161N)), xviii) SEQ ID NO:74 (IL-12p40(E73Q)), xix) SEQ IDNO:75 (IL-12p40(Q144E)), xx) SEQ ID NO:76 (IL-12p40(E262Q)), xxi) SEQ IDNO:77 (IL-12p40(E100Q)), xxii) SEQ ID NO:78 (IL-12p40(D18N)), xxiii) SEQID NO:79 (IL-12p40(E33Q)), xxiv) SEQ ID NO:80 (IL-12p40(Q229E)), xxv)SEQ ID NO:81 (IL-12p40(E235Q)), xxvi) SEQ ID NO:82 (IL-12p40(Q256N)),xxvii) SEQ ID NO:83 (IL-12p40(E299Q)), xxviii) SEQ ID NO:84(IL-12p40(D87N)), xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi)IL-12p40(S43E), xxxii) IL-12p40(S43K), xxxiii) SEQ ID NO:379 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E)), xxxiv) SEQ ID NO:205(IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii)IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl)SEQ ID NO:206 (IL-12p40(E32Q/E59Q)), xli) SEQ ID NO:207(IL-12p40(D34N/E59Q)), xlii) SEQ ID NO:208 (IL-12p40(E59Q/E187Q)),xliii) SEQ ID NO:209 (IL-12p40(S43E/E59Q)), xliv) SEQ ID NO:210(IL-12p40(S43K/E49Q)), xlv) SEQ ID NO:211 (IL-12p40(E59Q/K163E)), xlvi)SEQ ID NO:212 (IL-12p40(E59Q/K99E)), xlvii) SEQ ID NO:213(IL-12p40(E59Q/K258E)), xlviii) SEQ ID NO:214 (IL-12p40(E59Q/K260E)),xlix) SEQ ID NO: 326 (IL-12p40 (D34N/E59K)), 1) SEQ ID NO: 325(IL-12p40(E59K/K99E)), li) SEQ ID NO: 339 (IL-12p40(D18K/E59K/K99E)),lii) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), liii) SEQ ID NO: 336(IL-12p40 (E59K/K99Y)), liv) SEQ ID NO: 335 (IL-12p40 (E59Y/K99E)), lv)SEQ ID NO: 338 (IL-12p40 (E45K/E59K/K99E)), lvi) SEQ ID NO: 340(IL-12p40 (E59K/K99E/Q144E)), lvii) SEQ ID NO: 341 (IL-12p40(E59K/K99E/Q144K)), lviii) SEQ ID NO: 342 (IL-12p40 (E59K/K99E/R159E)),lix) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), lx) SEQ ID NO: 344(IL-12p40 (D18K/E59K/K99E/K264E)), lxi) SEQ ID NO: 360 (IL-12p40(C252S)), lxii) SEQ ID NO: 361 (IL-12p40 (DI8K/E59K/K99E/C252S)), lxiii)SEQ ID NO: 362 (IL-12p40 (D18K/E59K/K99E/C252S/K264E)), lxiv) SEQ ID NO:363 (IL-12p40 (E59K/K99Y/C252S)), lxv) SEQ ID NO: 364 (IL-12p40(E59K/K99E/C252S/K264E)), lxvi) SEQ ID NO: 365 (IL-12p40(E59K/K99E/C252S)), lxvii) SEQ ID NO: 254 (IL-12p40 (N103D/N113D)),lxviii) SEQ ID NO: 255 (IL-12p40 (N103D/N200D)), lxix) SEQ ID NO: 256(IL-12p40 (N103D/N281D)), lxx) SEQ ID NO: 257 (IL-12p40 (N113D/N200D)),lxxi) SEQ ID NO: 258 (IL-12p40 (N113D/N281D)), lxxii) SEQ ID NO: 259(IL-12p40 (N200D/N281D)), lxxiii) SEQ ID NO: 260 (IL-12p40(N103D/N113D/N200D)), lxxiv) SEQ ID NO: 261 (IL-12p40(N103D/N113D/N281D)), lxxv) SEQ ID NO: 262 (IL-12p40(N103D/N200D/N281D)), lxxvi) SEQ ID NO: 263 (IL-12p40(N113D/N200D/N281D)), lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)),lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)), lxxix) SEQ ID NO: 266(IL-12p40 (N200Q)), lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)), lxxxi) SEQID NO: 268 (IL-12p40 (N103Q/N113Q)), lxxxii) SEQ ID NO: 269 (IL-12p40(N103Q/N200Q)), lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q/N281Q)),lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q/N200Q)), lxxxv) SEQ ID NO: 272(IL-12p40 (N113Q/N281Q)), lxxxvi) SEQ ID NO: 273 (IL-12p40(N200Q/N281Q)), lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q/N113Q/N200Q)),lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q/N113Q/N281Q)), lxxxix) SEQ IDNO: 276 (IL-12p40 (N103Q/N200Q/N281Q)), xc) SEQ ID NO: 277 (IL-12p40(N113Q/N200Q/N281Q)), xci) SEQ ID NO:278 (IL-12p40(N103Q/N113Q/N200Q/N281Q)), xcii) SEQ ID NO:327 (IL-12p40(D34N/E59K/K99E)), xciii) SEQ ID NO:328 (IL-12p40 (D34K/E59K/K99E)),xciv) SEQ ID NO:329 (IL-12p40 (E32Q/D34N/E59K/K99E)), xcv) SEQ ID NO:331(IL-12p40 (E32K/D34N/E59K/K99E)), xcvi) SEQ ID NO: 337 (IL-12p40(E59Y/K99Y)), xcvii) SEQ ID NO:366 (IL-12p40(E59K/K99E/N103Q/C252S/K264E)), xcviii) SEQ ID NO:367 (IL-12p40(E59K/K99E/N113Q/C252S/K264E)), xcix) SEQ ID NO:368 (IL-12p40(E59K/K99E/N200Q/C252S/K264E)), c) SEQ ID NO:369 (IL-12p40(E59K/K99E/N281Q/C252S/K264E)), ci) SEQ ID NO:370 (IL-12p40(E59K/K99E/N103Q/N113Q/C252S/K264E)), cii) SEQ ID NO:371 (IL-12p40(E59K/K99E/N103Q/N200Q/C252S/K264E)), ciii) SEQ ID NO:372 (IL-12p40(E59K/K99E/N103Q/N281Q/C252S/K264E)), civ) SEQ ID NO:373 (IL-12p40(E59K/K99E/N113Q/N200Q/C252S/K264E)), cv) SEQ ID NO:374 (IL-12p40(E59K/K99E/N113Q/N281Q/C252S/K264E)), cvi) SEQ ID NO:375 (IL-12p40(E59K/K99E/N200Q/N281Q/C252S/K264E)), cvii) SEQ ID NO:376 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E)), cviii) SEQ ID NO:377(IL-12p40 (E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E)), and cix) SEQ IDNO:378 (IL-12p40 (E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E)).

In some embodiments, the present invention provides a heterodimericprotein wherein said IL-12p35 subunit has a sequence selected from thegroup consisting of: i) SEQ ID NO:113 (IL-12p35(N71D)), ii) SEQ IDNO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115 (IL-12p35(N195D)), iv) SEQID NO:116 (IL-12p35(N71D/N85D/N195D)), v) SEQ ID NO:117(IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii) SEQ IDNO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(Q135E)), ix) SEQID NO:121 (IL-12p35(Q35D)), x) SEQ ID NO:122 (IL-12p35(Q146E)), xi) SEQID NO:123 (IL-12p35(N76D)), xii) SEQ ID NO:124 (IL-12p35(E162Q)), xiii)SEQ ID NO:125 (IL-12p35(E163Q)), xiv) IL-12p35(N21D), xv) SEQ ID NO:333(IL-12p35(D55Q)), xvi) IL-12p35(E79Q), xvii) IL-12p35(Q130E), xviii)IL-12p35(N136D), xix) IL-12p35(E143Q), xx) SEQ ID NO:227(IL-12p35(N151K)), xxi) SEQ ID NO:226 (IL-12p35(E153K)), xxii)(IL-12p35(K158E)), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO:225(IL-12p35(N151D/E153Q)), xxv) SEQ ID NO:228 (IL-12p35(N151D/D165N)),xxvi) SEQ ID NO:229 (IL-12p35(Q130E/N151D)), xxvii) SEQ ID NO:230(IL-12p35(N151D/K158E)), xxviii) SEQ ID NO:231 (IL-12p35(E79Q/N151D)),xxix) SEQ ID NO:232 (IL-12p35(D55Q/N151D)), xxx) SEQ ID NO:233(IL-12p35(N136D/N151D)), xxxi) SEQ ID NO:234 (IL-12p35(N21D/N151D)),xxxii) SEQ ID NO:235 (IL-12p35(E143Q/N151D)), xxxiii) SEQ ID NO: 345(IL-12p35(F96A)), xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)), xxxv) SEQ IDNO: 347 (IL-12p35(L89A)), xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)),xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)), xxxviii) SEQ ID NO: 350(IL-12p35(L75A)), xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)), xxxv) SEQ IDNO: 279 (IL-12p35 (N71Q)), xxxvi) SEQ ID NO: 280 (IL-12p35 (N85Q)),xxxvii) SEQ ID NO: 281 (IL-12p35 (N195Q)), xxxviii) SEQ ID NO: 282(IL-12p35 (N71Q/N85Q)), xxxix) SEQ ID NO: 283 (IL-12p35 (N71Q/N195Q)),lx) SEQ ID NO: 284 (IL-12p35 (N85Q/N195Q), lxi) SEQ ID NO: 285 (IL-12p35(N71Q/N85Q/N195Q)), lxii) SEQ ID NO: 286 (IL-12p35 (N71D/N85D)), lxiii)SEQ ID NO: 287 (IL-12p35 (N71D/N195D), lxiv) SEQ ID NO: 288 (IL-12p35(N85D/N195D)), lxv) SEQ ID NO: 333 (IL-12p35 (D55Q)), and lxvi) SEQ IDNO: 334 (IL-12p35 (D55K)).

In another aspect, the present invention comprises a heterodimericprotein comprising: a) a first Fc domain; and b) a fusion proteincomprising: i) a second Fc domain; ii) an IL-12p40 subunit domain; andiii) an IL-12p35 subunit domain; wherein said first and second Fcdomains a set of amino acid substitutions selected from the groupconsisting of L368D/K370S and S364K; L368D/K370S and S364K/E357L;L368D/K370S and S364K/E357Q; T411E/K360E/Q362E and D401K; L368E/K370Sand S364K; K370S and S364K/E357Q; T366S/L368A/Y407V and T366W; andT366S/L368A/Y407V/Y349C and T366W/S354C; according to EU numbering.

In some embodiments, the present invention provides a heterodimericprotein wherein said fusion protein comprises, from N- to C-terminal:IL-12p40 subunit domain-domain linker-IL-12p35 subunit domain-domainlinker-hinge-CH2-CH3.

In some embodiments, the present invention provides a heterodimericprotein wherein said fusion protein comprises, from N- to C-terminal:IL-12p35 subunit domain-domain linker-IL-12p40 subunit domain-domainlinker-hinge-CH2-CH3.

In some embodiments, the present invention provides a heterodimericprotein wherein said fusion protein comprises, from N- to C-terminal:hinge-CH2-CH3-domain linker-IL-12p35 subunit domain-domainlinker-IL-12p40 subunit domain.

In some embodiments, the present invention provides a heterodimericprotein wherein said fusion protein comprises, from N- to C-terminal:hinge-CH2-CH3-domain linker-IL-12p40 subunit domain-domainlinker-IL-12p35 subunit domain.

In some embodiments, the present invention provides a heterodimericprotein wherein said IL-12p40 subunit has a sequence selected from thegroup consisting of: i) SEQ ID NO:57 (IL-12p40(N103D)), ii) SEQ ID NO:58(IL-12p40(N113D)), iii) SEQ ID NO:59 (IL-12p40(N200D)), iv) SEQ ID NO:60(IL-12p40(N281D)), v) SEQ ID NO:61 (IL-12p40(N103D/N113D/N200D/N281D)),vi) SEQ ID NO:62 (IL-12p40(Q42E)), vii) SEQ ID NO:63 (IL-12p40(E45Q)),viii) SEQ ID NO:64 (IL-12p40(Q56E)), ix) SEQ ID NO:65 (IL-12p40(E59Q)),x) SEQ ID NO:66 (IL-12p40(D62N)), xi) SEQ ID NO:67(IL-12p40(Q42E/E45Q)), xii) SEQ ID NO:68 (IL-12p40(E45Q/Q56E)), xiii)SEQ ID NO:69 (IL-12p40(Q42E/E59Q)), xiv) SEQ ID NO:70(IL-12p40(Q56E/E59Q)), xv) SEQ ID NO:71 (IL-12p40(Q42E/E45Q/Q56E)), xvi)SEQ ID NO:72 (IL-12p40(E45Q/Q56E/E59Q)), xvii) SEQ ID NO:73(IL-12p40(D161N)), xviii) SEQ ID NO:74 (IL-12p40(E73Q)), xix) SEQ IDNO:75 (IL-12p40(Q144E)), xx) SEQ ID NO:76 (IL-12p40(E262Q)), xxi) SEQ IDNO:77 (IL-12p40(E100Q)), xxii) SEQ ID NO:78 (IL-12p40(D18N)), xxiii) SEQID NO:79 (IL-12p40(E33Q)), xxiv) SEQ ID NO:80 (IL-12p40(Q229E)), xxv)SEQ ID NO:81 (IL-12p40(E235Q)), xxvi) SEQ ID NO:82 (IL-12p40(Q256N)),xxvii) SEQ ID NO:83 (IL-12p40(E299Q)), xxviii) SEQ ID NO:84(IL-12p40(D87N)), xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi)IL-12p40(S43E), xxxii) IL-12p40(S43K), xxxiii) SEQ ID NO:379 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E)), xxxiv) SEQ ID NO:205(IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii)IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl)SEQ ID NO:206 (IL-12p40(E32Q/E59Q)), xli) SEQ ID NO:207(IL-12p40(D34N/E59Q)), xlii) SEQ ID NO:208 (IL-12p40(E59Q/E187Q)),xliii) SEQ ID NO:209 (IL-12p40(S43E/E59Q)), xliv) SEQ ID NO:210(IL-12p40(S43K/E49Q)), xlv) SEQ ID NO:211 (IL-12p40(E59Q/K163E)), xlvi)SEQ ID NO:212 (IL-12p40(E59Q/K99E)), xlvii) SEQ ID NO:213(IL-12p40(E59Q/K258E)), xlviii) SEQ ID NO:214 (IL-12p40(E59Q/K260E)),xlix) SEQ ID NO: 326 (IL-12p40 (D34N/E59K)), 1) SEQ ID NO: 325(IL-12p40(E59K/K99E)), li) SEQ ID NO: 339 (IL-12p40(D18K/E59K/K99E)),lii) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), liii) SEQ ID NO: 336(IL-12p40 (E59K/K99Y)), liv) SEQ ID NO: 335 (IL-12p40 (E59Y/K99E)), lv)SEQ ID NO: 338 (IL-12p40 (E45K/E59K/K99E)), lvi) SEQ ID NO: 340(IL-12p40 (E59K/K99E/Q144E)), lvii) SEQ ID NO: 341 (IL-12p40(E59K/K99E/Q144K)), lviii) SEQ ID NO: 342 (IL-12p40 (E59K/K99E/R159E)),lix) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), lx) SEQ ID NO: 344(IL-12p40 (D18K/E59K/K99E/K264E)), lxi) SEQ ID NO: 360 (IL-12p40(C252S)), lxii) SEQ ID NO: 361 (IL-12p40 (DI8K/E59K/K99E/C252S)), lxiii)SEQ ID NO: 362 (IL-12p40 (D18K/E59K/K99E/C252S/K264E)), lxiv) SEQ ID NO:363 (IL-12p40 (E59K/K99Y/C252S)), lxv) SEQ ID NO: 364 (IL-12p40(E59K/K99E/C252S/K264E)), lxvi) SEQ ID NO: 365 (IL-12p40(E59K/K99E/C252S)), lxvii) SEQ ID NO: 254 (IL-12p40 (N103D/N113D)),lxviii) SEQ ID NO: 255 (IL-12p40 (N103D/N200D)), lxix) SEQ ID NO: 256(IL-12p40 (N103D/N281D)), lxx) SEQ ID NO: 257 (IL-12p40 (N113D/N200D)),lxxi) SEQ ID NO: 258 (IL-12p40 (N113D/N281D)), lxxii) SEQ ID NO: 259(IL-12p40 (N200D/N281D)), lxxiii) SEQ ID NO: 260 (IL-12p40(N103D/N113D/N200D)), lxxiv) SEQ ID NO: 261 (IL-12p40(N103D/N113D/N281D)), lxxv) SEQ ID NO: 262 (IL-12p40(N103D/N200D/N281D)), lxxvi) SEQ ID NO: 263 (IL-12p40(N113D/N200D/N281D)), lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)),lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)), lxxix) SEQ ID NO: 266(IL-12p40 (N200Q)), lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)), lxxxi) SEQID NO: 268 (IL-12p40 (N103Q/N113Q)), lxxxii) SEQ ID NO: 269 (IL-12p40(N103Q/N200Q)), lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q/N281Q)),lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q/N200Q)), lxxxv) SEQ ID NO: 272(IL-12p40 (N113Q/N281Q)), lxxxvi) SEQ ID NO: 273 (IL-12p40(N200Q/N281Q)), lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q/N113Q/N200Q)),lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q/N113Q/N281Q)), lxxxix) SEQ IDNO: 276 (IL-12p40 (N103Q/N200Q/N281Q)), xc) SEQ ID NO: 277 (IL-12p40(N113Q/N200Q/N281Q)), xci) SEQ ID NO:278 (IL-12p40(N103Q/N113Q/N200Q/N281Q)), xcii) SEQ ID NO:327 (IL-12p40(D34N/E59K/K99E)), xciii) SEQ ID NO:328 (IL-12p40 (D34K/E59K/K99E)),xciv) SEQ ID NO:329 (IL-12p40 (E32Q/D34N/E59K/K99E)), xcv) SEQ ID NO:331(IL-12p40 (E32K/D34N/E59K/K99E)), xcvi) SEQ ID NO: 337 (IL-12p40(E59Y/K99Y)), xcvii) SEQ ID NO:366 (IL-12p40(E59K/K99E/N103Q/C252S/K264E)), xcviii) SEQ ID NO:367 (IL-12p40(E59K/K99E/N113Q/C252S/K264E)), xcix) SEQ ID NO:368 (IL-12p40(E59K/K99E/N200Q/C252S/K264E)), c) SEQ ID NO:369 (IL-12p40(E59K/K99E/N281Q/C252S/K264E)), ci) SEQ ID NO:370 (IL-12p40(E59K/K99E/N103Q/N113Q/C252S/K264E)), cii) SEQ ID NO:371 (IL-12p40(E59K/K99E/N103Q/N200Q/C252S/K264E)), ciii) SEQ ID NO:372 (IL-12p40(E59K/K99E/N103Q/N281Q/C252S/K264E)), civ) SEQ ID NO:373 (IL-12p40(E59K/K99E/N113Q/N200Q/C252S/K264E)), cv) SEQ ID NO:374 (IL-12p40(E59K/K99E/N113Q/N281Q/C252S/K264E)), cvi) SEQ ID NO:375 (IL-12p40(E59K/K99E/N200Q/N281Q/C252S/K264E)), cvii) SEQ ID NO:376 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E)), cviii) SEQ ID NO:377(IL-12p40 (E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E)), and cix) SEQ IDNO:378 (IL-12p40 (E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E)).

In some embodiments, the present invention provides a heterodimericprotein wherein said IL-12p35 subunit has a sequence selected from thegroup consisting of: i) SEQ ID NO:113 (IL-12p35(N71D)), ii) SEQ IDNO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115 (IL-12p35(N195D)), iv) SEQID NO:116 (IL-12p35(N71D/N85D/N195D)), v) SEQ ID NO:117(IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii) SEQ IDNO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(Q135E)), ix) SEQID NO:121 (IL-12p35(Q35D)), x) SEQ ID NO:122 (IL-12p35(Q146E)), xi) SEQID NO:123 (IL-12p35(N76D)), xii) SEQ ID NO:124 (IL-12p35(E162Q)), xiii)SEQ ID NO:125 (IL-12p35(E163Q)), xiv) IL-12p35(N21D), xv) SEQ ID NO:333(IL-12p35(D55Q)), xvi) IL-12p35(E79Q), xvii) IL-12p35(Q130E), xviii)IL-12p35(N136D), xix) IL-12p35(E143Q), xx) SEQ ID NO:227(IL-12p35(N151K)), xxi) SEQ ID NO:226 (IL-12p35(E153K)), xxii)IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO:225(IL-12p35(N151D/E153Q)), xxv) SEQ ID NO:228 (IL-12p35(N151D/D165N)),xxvi) SEQ ID NO:229 (IL-12p35(Q130E/N151D)), xxvii) SEQ ID NO:230(IL-12p35(N151D/K158E)), xxviii) SEQ ID NO:231 (IL-12p35(E79Q/N151D)),xxix) SEQ ID NO:232 (IL-12p35(D55Q/N151D)), xxx) SEQ ID NO:233(IL-12p35(N136D/N151D)), xxxi) SEQ ID NO:234 (IL-12p35(N21D/N151D)),xxxii) SEQ ID NO:235 (IL-12p35(E143Q/N151D)), xxxiii) SEQ ID NO: 345(IL-12p35(F96A)), xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)), xxxv) SEQ IDNO: 347 (IL-12p35(L89A)), xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)),xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)), xxxviii) SEQ ID NO: 350(IL-12p35(L75A)), xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)), xxxv) SEQ IDNO: 279 (IL-12p35 (N71Q)), xxxvi) SEQ ID NO: 280 (IL-12p35 (N85Q)),xxxvii) SEQ ID NO: 281 (IL-12p35 (N195Q)), xxxviii) SEQ ID NO: 282(IL-12p35 (N71Q/N85Q)), xxxix) SEQ ID NO: 283 (IL-12p35 (N71Q/N195Q)),lx) SEQ ID NO: 284 (IL-12p35 (N85Q/N195Q), lxi) SEQ ID NO: 285 (IL-12p35(N71Q/N85Q/N195Q)), lxii) SEQ ID NO: 286 (IL-12p35 (N71D/N85D)), lxiii)SEQ ID NO: 287 (IL-12p35 (N71D/N195D), lxiv) SEQ ID NO: 288 (IL-12p35(N85D/N195D)), lxv) SEQ ID NO: 333 (IL-12p35 (D55Q)), and lxvi) SEQ IDNO: 334 (IL-12p35 (D55K)).

In another aspect, the present invention provides a homodimeric Fcfusion protein comprising a first monomer and a second monomer eachcomprising, from N- to C-terminal, an IL-12p40 subunit domain-anoptional first domain linker-an IL-12p35 subunit domain-an optionalsecond domain linker-an Fc domain.

In some embodiments, the present invention provides a homodimeric Fcfusion protein, wherein said modifications promoting homodimerization ofsaid Fc domain are a set of amino acid substitutions selected from thegroup consisting of L368D/K370S; S364K; S364K/E357L; S364K/E357Q;T411E/K360E/Q362E; D401K; T366S/L368A/Y407V; T366W;T366S/L368A/Y407V/Y349C; and T366W/S354C, according to EU numbering.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said first domain linker and said second domainlinker have the same amino acid sequence.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said Fc domain has an additional set of aminoacid substitutions comprising Q295E/N384D/Q418E/N421D, according to EUnumbering.

In some embodiments, the present invention provides a homodimeric Fcfusion protein, wherein said Fc domain has an additional set of aminoacid substitutions selected from the group consisting of G236R/L328R,E233P/L234V/L235A/G236_/S239K, E233P/L234V/L235A/G236_/S239K/A327G,E233P/L234V/L235A/G236_/S267K/A327G, E233P/L234V/L235A/G236_, andE233P/L234V/L235A/G236_/S267K, according to EU numbering.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected form the group consisting of SEQ ID NO: 3 (human IL-12 subunitbeta (IL-12p40) precursor sequence) and SEQ ID NO:4 (human IL-12 subunitbeta (IL-12p40) mature form sequence), and said IL-12p35 subunit has apolypeptide sequence selected from the group consisting of SEQ ID NO:1(human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ IDNO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein each of said Fc domain further comprises aminoacid substitutions M428L/N434S.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit and/or said IL-12p35 subunit is a variant IL-12p35 subunit.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex; and/or said IL-12p35 subunit is a variant IL-12p35subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidmodifications at amino acid residues selected from the group consistingof E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32,E33, D34, L40, D41, Q42, S43, E45, L47, T54, 155, Q56, K58, E59, F60,G61, D62, Q65, Y66, E73, K84, E86, D87, G88, 189, W90, D93, D97, K99,E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156,R159, D161, N162, K163, D166, D170, Q172, D174, A176, C177, P178, A179,A180, E181, S183, P185, E187, N200, S204, F206, R208, D209, D214, N218,Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F247, S248, C252S,Q256, K158, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292,Y293, and E299.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidsubstitutions selected from the group consisting of D18N, D18K, E32Q,E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q,D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N,R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N,K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has amino acidsubstitutions selected from the group consisting ofN103D/N113D/N200D/N281D, Q42E/E45Q, E45Q/Q56E, Q42E/E59Q, Q56E/E59Q,Q42E/E45Q/Q56E, E45Q/Q56E/E59Q, E32Q/E59Q, D34N/E59K, D34N/E59K/K99E,D34K/E59K/K99E, E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q,E59Q/E187Q, S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E,E59Q/K260E, E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y,E59Y/K99Y, E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:57(IL-12p40(N103D)), ii) SEQ ID NO:58 (IL-12p40(N113D)), iii) SEQ ID NO:59(IL-12p40(N200D)), iv) SEQ ID NO:60 (IL-12p40(N281D)), v) SEQ ID NO:61(IL-12p40(N103D/N113D/N200D/N281D)), vi) SEQ ID NO:62 (IL-12p40(Q42E)),vii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)),ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi)SEQ ID NO:67 (IL-12p40(Q42E/E45Q)), xii) SEQ ID NO:68(IL-12p40(E45Q/Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E/E59Q)), xiv)SEQ ID NO:70 (IL-12p40(Q56E/E59Q)), xv) SEQ ID NO:71(IL-12p40(Q42E/E45Q/Q56E)), xvi) SEQ ID NO:72(IL-12p40(E45Q/Q56E/E59Q)), xvii) SEQ ID NO:73 (IL-12p40(D161N)), xviii)SEQ ID NO:74 (IL-12p40(E73Q)), xix) SEQ ID NO:75 (IL-12p40(Q144E)), xx)SEQ ID NO:76 (IL-12p40(E262Q)), xxi) SEQ ID NO:77 (IL-12p40(E100Q)),xxii) SEQ ID NO:78 (IL-12p40(D18N)), xxiii) SEQ ID NO:79(IL-12p40(E33Q)), xxiv) SEQ ID NO:80 (IL-12p40(Q229E)), xxv) SEQ IDNO:81 (IL-12p40(E235Q)), xxvi) SEQ ID NO:82 (IL-12p40(Q256N)), xxvii)SEQ ID NO:83 (IL-12p40(E299Q)), xxviii) SEQ ID NO:84 (IL-12p40(D87N)),xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii)IL-12p40(S43K), xxxiii) SEQ ID NO:379 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E)), xxxiv) SEQ ID NO:205(IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii)IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl)SEQ ID NO:206 (IL-12p40(E32Q/E59Q)), xli) SEQ ID NO:207(IL-12p40(D34N/E59Q)), xlii) SEQ ID NO:208 (IL-12p40(E59Q/E187Q)),xliii) SEQ ID NO:209 (IL-12p40(S43E/E59Q)), xliv) SEQ ID NO:210(IL-12p40(S43K/E49Q)), xlv) SEQ ID NO:211 (IL-12p40(E59Q/K163E)), xlvi)SEQ ID NO:212 (IL-12p40(E59Q/K99E)), xlvii) SEQ ID NO:213(IL-12p40(E59Q/K258E)), xlviii) SEQ ID NO:214 (IL-12p40(E59Q/K260E)),xlix) SEQ ID NO: 326 (IL-12p40 (D34N/E59K)), 1) SEQ ID NO: 325(IL-12p40(E59K/K99E)), li) SEQ ID NO: 339 (IL-12p40(D18K/E59K/K99E)),lii) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), liii) SEQ ID NO: 336(IL-12p40 (E59K/K99Y)), liv) SEQ ID NO: 335 (IL-12p40 (E59Y/K99E)), lv)SEQ ID NO: 338 (IL-12p40 (E45K/E59K/K99E)), lvi) SEQ ID NO: 340(IL-12p40 (E59K/K99E/Q144E)), lvii) SEQ ID NO: 341 (IL-12p40(E59K/K99E/Q144K)), lviii) SEQ ID NO: 342 (IL-12p40 (E59K/K99E/R159E)),lix) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), lx) SEQ ID NO: 344(IL-12p40 (D18K/E59K/K99E/K264E)), lxi) SEQ ID NO: 360 (IL-12p40(C252S)), lxii) SEQ ID NO: 361 (IL-12p40 (DI8K/E59K/K99E/C252S)), lxiii)SEQ ID NO: 362 (IL-12p40 (D18K/E59K/K99E/C252S/K264E)), lxiv) SEQ ID NO:363 (IL-12p40 (E59K/K99Y/C252S)), lxv) SEQ ID NO: 364 (IL-12p40(E59K/K99E/C252S/K264E)), lxvi) SEQ ID NO: 365 (IL-12p40(E59K/K99E/C252S)), lxvii) SEQ ID NO: 254 (IL-12p40 (N103D/N113D)),lxviii) SEQ ID NO: 255 (IL-12p40 (N103D/N200D)), lxix) SEQ ID NO: 256(IL-12p40 (N103D/N281D)), lxx) SEQ ID NO: 257 (IL-12p40 (N113D/N200D)),lxxi) SEQ ID NO: 258 (IL-12p40 (N113D/N281D)), lxxii) SEQ ID NO: 259(IL-12p40 (N200D/N281D)), lxxiii) SEQ ID NO: 260 (IL-12p40(N103D/N113D/N200D)), lxxiv) SEQ ID NO: 261 (IL-12p40(N103D/N113D/N281D)), lxxv) SEQ ID NO: 262 (IL-12p40(N103D/N200D/N281D)), lxxvi) SEQ ID NO: 263 (IL-12p40(N113D/N200D/N281D)), lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)),lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)), lxxix) SEQ ID NO: 266(IL-12p40 (N200Q)), lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)), lxxxi) SEQID NO: 268 (IL-12p40 (N103Q/N113Q)), lxxxii) SEQ ID NO: 269 (IL-12p40(N103Q/N200Q)), lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q/N281Q)),lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q/N200Q)), lxxxv) SEQ ID NO: 272(IL-12p40 (N113Q/N281Q)), lxxxvi) SEQ ID NO: 273 (IL-12p40(N200Q/N281Q)), lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q/N113Q/N200Q)),lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q/N113Q/N281Q)), lxxxix) SEQ IDNO: 276 (IL-12p40 (N103Q/N200Q/N281Q)), xc) SEQ ID NO: 277 (IL-12p40(N113Q/N200Q/N281Q)), xci) SEQ ID NO:278 (IL-12p40(N103Q/N113Q/N200Q/N281Q)), xcii) SEQ ID NO:327 (IL-12p40(D34N/E59K/K99E)), xciii) SEQ ID NO:328 (IL-12p40 (D34K/E59K/K99E)),xciv) SEQ ID NO:329 (IL-12p40 (E32Q/D34N/E59K/K99E)), xcv) SEQ ID NO:331(IL-12p40 (E32K/D34N/E59K/K99E)), xcvi) SEQ ID NO: 337 (IL-12p40(E59Y/K99Y)), xcvii) SEQ ID NO:366 (IL-12p40(E59K/K99E/N103Q/C252S/K264E)), xcviii) SEQ ID NO:367 (IL-12p40(E59K/K99E/N113Q/C252S/K264E)), xcix) SEQ ID NO:368 (IL-12p40(E59K/K99E/N200Q/C252S/K264E)), c) SEQ ID NO:369 (IL-12p40(E59K/K99E/N281Q/C252S/K264E)), ci) SEQ ID NO:370 (IL-12p40(E59K/K99E/N103Q/N113Q/C252S/K264E)), cii) SEQ ID NO:371 (IL-12p40(E59K/K99E/N103Q/N200Q/C252S/K264E)), ciii) SEQ ID NO:372 (IL-12p40(E59K/K99E/N103Q/N281Q/C252S/K264E)), civ) SEQ ID NO:373 (IL-12p40(E59K/K99E/N113Q/N200Q/C252S/K264E)), cv) SEQ ID NO:374 (IL-12p40(E59K/K99E/N113Q/N281Q/C252S/K264E)), cvi) SEQ ID NO:375 (IL-12p40(E59K/K99E/N200Q/N281Q/C252S/K264E)), cvii) SEQ ID NO:376 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E)), cviii) SEQ ID NO:377(IL-12p40 (E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E)), and cix) SEQ IDNO:378 (IL-12p40 (E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E)).

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidmodifications as amino acid residues selected from the group consistingof Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63,L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97,L124, M125, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163,D165, I171, R181, 1182, R183, V185, T186, D188, R189, V190, S192, Y193,N195, and A196.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidsubstitutions selected from the group consisting of N21D, Q35D, E38Q,D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A,L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K,E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has amino acidsubstitutions selected from the group consisting of N71D/N85D/N195D,N151D/E153Q, N151D/D165N, Q130E/N151D, N151D/K158E, E79Q/N151D,D55Q/N151D, N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q, N71Q/N195Q,N85Q/N195Q, N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, and N85D/N195D.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:113(IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115(IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D/N85D/N195D)), v) SEQID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii)SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(Q135E)),ix) SEQ ID NO:121 (IL-12p35(Q35D)), x) SEQ ID NO:122 (IL-12p35(Q146E)),xi) SEQ ID NO:123 (IL-12p35(N76D)), xii) SEQ ID NO:124(IL-12p35(E162Q)), xiii) SEQ ID NO:125 (IL-12p35(E163Q)), xiv)IL-12p35(N21D), xv) SEQ ID NO:333 (IL-12p35(D55Q)), xvi) IL-12p35(E79Q),xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx)SEQ ID NO:227 (IL-12p35(N151K)), xxi) SEQ ID NO:226 (IL-12p35(E153K)),xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO:225(IL-12p35(N151D/E153Q)), xxv) SEQ ID NO:228 (IL-12p35(N151D/D165N)),xxvi) SEQ ID NO:229 (IL-12p35(Q130E/N151D)), xxvii) SEQ ID NO:230(IL-12p35(N151D/K158E)), xxviii) SEQ ID NO:231 (IL-12p35(E79Q/N151D)),xxix) SEQ ID NO:232 (IL-12p35(D55Q/N151D)), xxx) SEQ ID NO:233(IL-12p35(N136D/N151D)), xxxi) SEQ ID NO:234 (IL-12p35(N21D/N151D)),xxxii) SEQ ID NO:235 (IL-12p35(E143Q/N151D)), xxxiii) SEQ ID NO: 345(IL-12p35(F96A)), xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)), xxxv) SEQ IDNO: 347 (IL-12p35(L89A)), xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)),xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)), xxxviii) SEQ ID NO: 350(IL-12p35(L75A)), xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)), xxxv) SEQ IDNO: 279 (IL-12p35 (N71Q)), xxxvi) SEQ ID NO: 280 (IL-12p35 (N85Q)),xxxvii) SEQ ID NO: 281 (IL-12p35 (N195Q)), xxxviii) SEQ ID NO: 282(IL-12p35 (N71Q/N85Q)), xxxix) SEQ ID NO: 283 (IL-12p35 (N71Q/N195Q)),xl) SEQ ID NO: 284 (IL-12p35 (N85Q/N195Q), xli) SEQ ID NO: 285 (IL-12p35(N71Q/N85Q/N195Q)), xlii) SEQ ID NO: 286 (IL-12p35 (N71D/N85D)), xliii)SEQ ID NO: 287 (IL-12p35 (N71D/N195D), xliv) SEQ ID NO: 288 (IL-12p35(N85D/N195D)), xlv) SEQ ID NO: 333 (IL-12p35 (D55Q)), and xlvi) SEQ IDNO: 334 (IL-12p35 (D55K)).

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said first and second monomers are XENP31289.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said first and second monomers are XENP31291.

In some embodiments, the present invention provides a homodimeric Fcfusion protein composition comprising a homodimeric Fc fusion proteinfor use in treating cancer in a subject.

In some embodiments, the present invention provides a one or morenucleic acids encoding a homodimeric Fc fusion protein.

In some embodiments, the present invention provides a host cellcomprising said one or more nucleic acids encoding a homodimeric Fcfusion protein.

In some embodiments, the present invention provides a method of making ahomodimeric Fc fusion protein, said method comprising culturing a hostcell under conditions whereby said homodimeric Fc fusion protein isproduced.

In some embodiments, the present invention provides a method ofpurifying a homodimeric Fc fusion protein, said method comprising: a)providing a composition comprising the homodimeric Fc fusion protein; b)loading said composition onto an ion exchange column; and c) collectinga fraction containing said homodimeric Fc fusion protein.

In another aspect, the present invention provides a homodimeric Fcfusion protein comprising a first monomer and a second monomer eachcomprising, from N- to C-terminal, an IL-12p35 subunit domain-anoptional first domain linker-an IL-12p40 subunit domain-an optionalsecond domain linker-an Fc domain.

In some embodiments, the present invention provides a homodimeric Fcfusion protein, wherein said modifications promoting homodimerization ofsaid Fc domain are a set of amino acid substitutions selected from thegroup consisting of L368D/K370S; S364K; S364K/E357L; S364K/E357Q;T411E/K360E/Q362E; D401K; T366S/L368A/Y407V; T366W;T366S/L368A/Y407V/Y349C; and T366W/S354C, according to EU numbering.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said first domain linker and said second domainlinker have the same amino acid sequence.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said Fc domain has an additional set of aminoacid substitutions comprising Q295E/N384D/Q418E/N421D, according to EUnumbering.

In some embodiments, the present invention provides a homodimeric Fcfusion protein, wherein said Fc domain has an additional set of aminoacid substitutions selected from the group consisting of G236R/L328R,E233P/L234V/L235A/G236_/S239K, E233P/L234V/L235A/G236_/S239K/A327G,E233P/L234V/L235A/G236_/S267K/A327G, E233P/L234V/L235A/G236_, andE233P/L234V/L235A/G236_/S267K, according to EU numbering.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected form the group consisting of SEQ ID NO:3 (human IL-12 subunitbeta (IL-12p40) precursor sequence) and SEQ ID NO:4 (human IL-12 subunitbeta (IL-12p40) mature form sequence), and said IL-12p35 subunit has apolypeptide sequence selected from the group consisting of SEQ ID NO:1(human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ IDNO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein each of said Fc domain further comprises aminoacid substitutions M428L/N434S.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit and/or said IL-12p35 subunit is a variant IL-12p35 subunit.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex; and/or said IL-12p35 subunit is a variant IL-12p35subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidmodifications at amino acid residues selected from the group consistingof E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32,E33, D34, L40, D41, Q42, S43, E45, L47, T54, 155, Q56, K58, E59, F60,G61, D62, Q65, Y66, E73, K84, E86, D87, G88, 189, W90, D93, D97, K99,E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156,R159, D161, N162, K163, D166, D170, Q172, D174, A176, C177, P178, A179,A180, E181, S183, P185, E187, N200, S204, F206, R208, D209, D214, N218,Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F247, S248, C252S,Q256, K158, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292,Y293, and E299.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidsubstitutions selected from the group consisting of D18N, D18K, E32Q,E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q,D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N,R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N,K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has amino acidsubstitutions selected from the group consisting ofN103D/N113D/N200D/N281D, Q42E/E45Q, E45Q/Q56E, Q42E/E59Q, Q56E/E59Q,Q42E/E45Q/Q56E, E45Q/Q56E/E59Q, E32Q/E59Q, D34N/E59K, D34N/E59K/K99E,D34K/E59K/K99E, E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q,E59Q/E187Q, S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E,E59Q/K260E, E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y,E59Y/K99Y, E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected from the group consisting of:

i) SEQ ID NO:57 (IL-12p40(N103D)), ii) SEQ ID NO:58 (IL-12p40(N113D)),iii) SEQ ID NO:59 (IL-12p40(N200D)), iv) SEQ ID NO:60 (IL-12p40(N281D)),v) SEQ ID NO:61 (IL-12p40(N103D/N113D/N200D/N281D)), vi) SEQ ID NO:62(IL-12p40(Q42E)), vii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64(IL-12p40(Q56E)), ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66(IL-12p40(D62N)), xi) SEQ ID NO:67 (IL-12p40(Q42E/E45Q)), xii) SEQ IDNO:68 (IL-12p40(E45Q/Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E/E59Q)),xiv) SEQ ID NO:70 (IL-12p40(Q56E/E59Q)), xv) SEQ ID NO:71(IL-12p40(Q42E/E45Q/Q56E)), xvi) SEQ ID NO:72(IL-12p40(E45Q/Q56E/E59Q)), xvii) SEQ ID NO:73 (IL-12p40(D161N)), xviii)SEQ ID NO:74 (IL-12p40(E73Q)), xix) SEQ ID NO:75 (IL-12p40(Q144E)), xx)SEQ ID NO:76 (IL-12p40(E262Q)), xxi) SEQ ID NO:77 (IL-12p40(E100Q)),xxii) SEQ ID NO:78 (IL-12p40(D18N)), xxiii) SEQ ID NO:79(IL-12p40(E33Q)), xxiv) SEQ ID NO:80 (IL-12p40(Q229E)), xxv) SEQ IDNO:81 (IL-12p40(E235Q)), xxvi) SEQ ID NO:82 (IL-12p40(Q256N)), xxvii)SEQ ID NO:83 (IL-12p40(E299Q)), xxviii) SEQ ID NO:84 (IL-12p40(D87N)),xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii)IL-12p40(S43K), xxxiii) SEQ ID NO:379 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E)), xxxiv) SEQ ID NO:205(IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii)IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl)SEQ ID NO:206 (IL-12p40(E32Q/E59Q)), xli) SEQ ID NO:207(IL-12p40(D34N/E59Q)), xlii) SEQ ID NO:208 (IL-12p40(E59Q/E187Q)),xliii) SEQ ID NO:209 (IL-12p40(S43E/E59Q)), xliv) SEQ ID NO:210(IL-12p40(S43K/E49Q)), xlv) SEQ ID NO:211 (IL-12p40(E59Q/K163E)), xlvi)SEQ ID NO:212 (IL-12p40(E59Q/K99E)), xlvii) SEQ ID NO:213(IL-12p40(E59Q/K258E)), xlviii) SEQ ID NO:214 (IL-12p40(E59Q/K260E)),xlix) SEQ ID NO: 326 (IL-12p40 (D34N/E59K)), 1) SEQ ID NO: 325(IL-12p40(E59K/K99E)), li) SEQ ID NO: 339 (IL-12p40(D18K/E59K/K99E)),lii) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), liii) SEQ ID NO: 336(IL-12p40 (E59K/K99Y)), liv) SEQ ID NO: 335 (IL-12p40 (E59Y/K99E)), lv)SEQ ID NO: 338 (IL-12p40 (E45K/E59K/K99E)), lvi) SEQ ID NO: 340(IL-12p40 (E59K/K99E/Q144E)), lvii) SEQ ID NO: 341 (IL-12p40(E59K/K99E/Q144K)), lviii) SEQ ID NO: 342 (IL-12p40 (E59K/K99E/R159E)),lix) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), lx) SEQ ID NO: 344(IL-12p40 (D18K/E59K/K99E/K264E)), lxi) SEQ ID NO: 360 (IL-12p40(C252S)), lxii) SEQ ID NO: 361 (IL-12p40 (DI8K/E59K/K99E/C252S)), lxiii)SEQ ID NO: 362 (IL-12p40 (D18K/E59K/K99E/C252S/K264E)), lxiv) SEQ ID NO:363 (IL-12p40 (E59K/K99Y/C252S)), lxv) SEQ ID NO: 364 (IL-12p40(E59K/K99E/C252S/K264E)), lxvi) SEQ ID NO: 365 (IL-12p40(E59K/K99E/C252S)), lxvii) SEQ ID NO: 254 (IL-12p40 (N103D/N113D)),lxviii) SEQ ID NO: 255 (IL-12p40 (N103D/N200D)), lxix) SEQ ID NO: 256(IL-12p40 (N103D/N281D)), lxx) SEQ ID NO: 257 (IL-12p40 (N113D/N200D)),lxxi) SEQ ID NO: 258 (IL-12p40 (N113D/N281D)), lxxii) SEQ ID NO: 259(IL-12p40 (N200D/N281D)), lxxiii) SEQ ID NO: 260 (IL-12p40(N103D/N113D/N200D)), lxxiv) SEQ ID NO: 261 (IL-12p40(N103D/N113D/N281D)), lxxv) SEQ ID NO: 262 (IL-12p40(N103D/N200D/N281D)), lxxvi) SEQ ID NO: 263 (IL-12p40(N113D/N200D/N281D)), lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)),lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)), lxxix) SEQ ID NO: 266(IL-12p40 (N200Q)), lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)), lxxxi) SEQID NO: 268 (IL-12p40 (N103Q/N113Q)), lxxxii) SEQ ID NO: 269 (IL-12p40(N103Q/N200Q)), lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q/N281Q)),lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q/N200Q)), lxxxv) SEQ ID NO: 272(IL-12p40 (N113Q/N281Q)), lxxxvi) SEQ ID NO: 273 (IL-12p40(N200Q/N281Q)), lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q/N113Q/N200Q)),lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q/N113Q/N281Q)), lxxxix) SEQ IDNO: 276 (IL-12p40 (N103Q/N200Q/N281Q)), xc) SEQ ID NO: 277 (IL-12p40(N113Q/N200Q/N281Q)), xci) SEQ ID NO:278 (IL-12p40(N103Q/N113Q/N200Q/N281Q)), xcii) SEQ ID NO:327 (IL-12p40(D34N/E59K/K99E)), xciii) SEQ ID NO:328 (IL-12p40 (D34K/E59K/K99E)),xciv) SEQ ID NO:329 (IL-12p40 (E32Q/D34N/E59K/K99E)), xcv) SEQ ID NO:331(IL-12p40 (E32K/D34N/E59K/K99E)), xcvi) SEQ ID NO: 337 (IL-12p40(E59Y/K99Y)), xcvii) SEQ ID NO:366 (IL-12p40(E59K/K99E/N103Q/C252S/K264E)), xcviii) SEQ ID NO:367 (IL-12p40(E59K/K99E/N113Q/C252S/K264E)), xcix) SEQ ID NO:368 (IL-12p40(E59K/K99E/N200Q/C252S/K264E)), c) SEQ ID NO:369 (IL-12p40(E59K/K99E/N281Q/C252S/K264E)), ci) SEQ ID NO:370 (IL-12p40(E59K/K99E/N103Q/N113Q/C252S/K264E)), cii) SEQ ID NO:371 (IL-12p40(E59K/K99E/N103Q/N200Q/C252S/K264E)), ciii) SEQ ID NO:372 (IL-12p40(E59K/K99E/N103Q/N281Q/C252S/K264E)), civ) SEQ ID NO:373 (IL-12p40(E59K/K99E/N113Q/N200Q/C252S/K264E)), cv) SEQ ID NO:374 (IL-12p40(E59K/K99E/N113Q/N281Q/C252S/K264E)), cvi) SEQ ID NO:375 (IL-12p40(E59K/K99E/N200Q/N281Q/C252S/K264E)), cvii) SEQ ID NO:376 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E)), cviii) SEQ ID NO:377(IL-12p40 (E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E)), and cix) SEQ IDNO:378 (IL-12p40 (E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E)).

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidmodifications as amino acid residues selected from the group consistingof Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63,L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97,L124, M125, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163,D165, I171, R181, 1182, R183, V185, T186, D188, R189, V190, S192, Y193,N195, and A196.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidsubstitutions selected from the group consisting of N21D, Q35D, E38Q,D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A,L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K,E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has amino acidsubstitutions selected from the group consisting of N71D/N85D/N195D,N151D/E153Q, N151D/D165N, Q130E/N151D, N151D/K158E, E79Q/N151D,D55Q/N151D, N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q, N71Q/N195Q,N85Q/N195Q, N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, and N85D/N195D.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:113(IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115(IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D/N85D/N195D)), v) SEQID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii)SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(Q135E)),ix) SEQ ID NO:121 (IL-12p35(Q35D)), x) SEQ ID NO:122 (IL-12p35(Q146E)),xi) SEQ ID NO:123 (IL-12p35(N76D)), xii) SEQ ID NO:124(IL-12p35(E162Q)), xiii) SEQ ID NO:125 (IL-12p35(E163Q)), xiv)IL-12p35(N21D), xv) SEQ ID NO:333 (IL-12p35(D55Q)), xvi) IL-12p35(E79Q),xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx)SEQ ID NO:227 (IL-12p35(N151K)), xxi) SEQ ID NO:226 (IL-12p35(E153K)),xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO:225(IL-12p35(N151D/E153Q)), xxv) SEQ ID NO:228 (IL-12p35(N151D/D165N)),xxvi) SEQ ID NO:229 (IL-12p35(Q130E/N151D)), xxvii) SEQ ID NO:230(IL-12p35(N151D/K158E)), xxviii) SEQ ID NO:231 (IL-12p35(E79Q/N151D)),xxix) SEQ ID NO:232 (IL-12p35(D55Q/N151D)), xxx) SEQ ID NO:233(IL-12p35(N136D/N151D)), xxxi) SEQ ID NO:234 (IL-12p35(N21D/N151D)),xxxii) SEQ ID NO:235 (IL-12p35(E143Q/N151D)), xxxiii) SEQ ID NO: 345(IL-12p35(F96A)), xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)), xxxv) SEQ IDNO: 347 (IL-12p35(L89A)), xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)),xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)), xxxviii) SEQ ID NO: 350(IL-12p35(L75A)), xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)), xxxv) SEQ IDNO: 279 (IL-12p35 (N71Q)), xxxvi) SEQ ID NO: 280 (IL-12p35 (N85Q)),xxxvii) SEQ ID NO: 281 (IL-12p35 (N195Q)), xxxviii) SEQ ID NO: 282(IL-12p35 (N71Q/N85Q)), xxxix) SEQ ID NO: 283 (IL-12p35 (N71Q/N195Q)),xl) SEQ ID NO: 284 (IL-12p35 (N85Q/N195Q), xli) SEQ ID NO: 285 (IL-12p35(N71Q/N85Q/N195Q)), xlii) SEQ ID NO: 286 (IL-12p35 (N71D/N85D)), xliii)SEQ ID NO: 287 (IL-12p35 (N71D/N195D), xliv) SEQ ID NO: 288 (IL-12p35(N85D/N195D)), xlv) SEQ ID NO: 333 (IL-12p35 (D55Q)), and xlvi) SEQ IDNO: 334 (IL-12p35 (D55K)).

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said first and second monomers are XENP31289.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said first and second monomers are XENP31291.

In some embodiments, the present invention provides a homodimeric Fcfusion protein composition comprising a homodimeric Fc fusion proteinfor use in treating cancer in a subject.

In some embodiments, the present invention provides a one or morenucleic acids encoding a homodimeric Fc fusion protein.

In some embodiments, the present invention provides a host cellcomprising said one or more nucleic acids encoding a homodimeric Fcfusion protein.

In some embodiments, the present invention provides a method of making ahomodimeric Fc fusion protein, said method comprising culturing a hostcell under conditions whereby said homodimeric Fc fusion protein isproduced.

In some embodiments, the present invention provides a method ofpurifying a homodimeric Fc fusion protein, said method comprising: a)providing a composition comprising the homodimeric Fc fusion protein; b)loading said composition onto an ion exchange column; and c) collectinga fraction containing said homodimeric Fc fusion protein.

In another aspect, the present invention provides a homodimeric Fcfusion protein comprising a first monomer and a second monomer eachcomprising, from N- to C-terminal, an Fc domain-an optional first domainlinker-an IL-12p40 subunit domain-an optional second domain linker-anIL-12p35 subunit domain.

In some embodiments, the present invention provides a homodimeric Fcfusion protein, wherein said modifications promoting homodimerization ofsaid Fc domain are a set of amino acid substitutions selected from thegroup consisting of L368D/K370S; S364K; S364K/E357L; S364K/E357Q;T411E/K360E/Q362E; D401K; T366S/L368A/Y407V; T366W;T366S/L368A/Y407V/Y349C; and T366W/S354C, according to EU numbering.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said first domain linker and said second domainlinker have the same amino acid sequence.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said Fc domain has an additional set of aminoacid substitutions comprising Q295E/N384D/Q418E/N421D, according to EUnumbering.

In some embodiments, the present invention provides a homodimeric Fcfusion protein, wherein said Fc domain has an additional set of aminoacid substitutions selected from the group consisting of G236R/L328R,E233P/L234V/L235A/G236_/S239K, E233P/L234V/L235A/G236_/S239K/A327G,E233P/L234V/L235A/G236_/S267K/A327G, E233P/L234V/L235A/G236_, andE233P/L234V/L235A/G236_/S267K, according to EU numbering.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected form the group consisting of SEQ ID NO:3 (human IL-12 subunitbeta (IL-12p40) precursor sequence) and SEQ ID NO:4 (human IL-12 subunitbeta (IL-12p40) mature form sequence), and said IL-12p35 subunit has apolypeptide sequence selected from the group consisting of SEQ ID NO:1(human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ IDNO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein each of said Fc domain further comprises aminoacid substitutions M428L/N434S.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit and/or said IL-12p35 subunit is a variant IL-12p35 subunit.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex; and/or said IL-12p35 subunit is a variant IL-12p35subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidmodifications at amino acid residues selected from the group consistingof E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32,E33, D34, L40, D41, Q42, S43, E45, L47, T54, 155, Q56, K58, E59, F60,G61, D62, Q65, Y66, E73, K84, E86, D87, G88, 189, W90, D93, D97, K99,E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156,R159, D161, N162, K163, D166, D170, Q172, D174, A176, C177, P178, A179,A180, E181, S183, P185, E187, N200, S204, F206, R208, D209, D214, N218,Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F247, S248, C252S,Q256, K158, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292,Y293, and E299.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidsubstitutions selected from the group consisting of D18N, D18K, E32Q,E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q,D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N,R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N,K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has amino acidsubstitutions selected from the group consisting ofN103D/N113D/N200D/N281D, Q42E/E45Q, E45Q/Q56E, Q42E/E59Q, Q56E/E59Q,Q42E/E45Q/Q56E, E45Q/Q56E/E59Q, E32Q/E59Q, D34N/E59K, D34N/E59K/K99E,D34K/E59K/K99E, E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q,E59Q/E187Q, S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E,E59Q/K260E, E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y,E59Y/K99Y, E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:57(IL-12p40(N103D)), ii) SEQ ID NO:58 (IL-12p40(N113D)), iii) SEQ ID NO:59(IL-12p40(N200D)), iv) SEQ ID NO:60 (IL-12p40(N281D)), v) SEQ ID NO:61(IL-12p40(N103D/N113D/N200D/N281D)), vi) SEQ ID NO:62 (IL-12p40(Q42E)),vii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)),ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi)SEQ ID NO:67 (IL-12p40(Q42E/E45Q)), xii) SEQ ID NO:68(IL-12p40(E45Q/Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E/E59Q)), xiv)SEQ ID NO:70 (IL-12p40(Q56E/E59Q)), xv) SEQ ID NO:71(IL-12p40(Q42E/E45Q/Q56E)), xvi) SEQ ID NO:72(IL-12p40(E45Q/Q56E/E59Q)), xvii) SEQ ID NO:73 (IL-12p40(D161N)), xviii)SEQ ID NO:74 (IL-12p40(E73Q)), xix) SEQ ID NO:75 (IL-12p40(Q144E)), xx)SEQ ID NO:76 (IL-12p40(E262Q)), xxi) SEQ ID NO:77 (IL-12p40(E100Q)),xxii) SEQ ID NO:78 (IL-12p40(D18N)), xxiii) SEQ ID NO:79(IL-12p40(E33Q)), xxiv) SEQ ID NO:80 (IL-12p40(Q229E)), xxv) SEQ IDNO:81 (IL-12p40(E235Q)), xxvi) SEQ ID NO:82 (IL-12p40(Q256N)), xxvii)SEQ ID NO:83 (IL-12p40(E299Q)), xxviii) SEQ ID NO:84 (IL-12p40(D87N)),xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii)IL-12p40(S43K), xxxiii) SEQ ID NO:379 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E)), xxxiv) SEQ ID NO:205(IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii)IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl)SEQ ID NO:206 (IL-12p40(E32Q/E59Q)), xli) SEQ ID NO:207(IL-12p40(D34N/E59Q)), xlii) SEQ ID NO:208 (IL-12p40(E59Q/E187Q)),xliii) SEQ ID NO:209 (IL-12p40(S43E/E59Q)), xliv) SEQ ID NO:210(IL-12p40(S43K/E49Q)), xlv) SEQ ID NO:211 (IL-12p40(E59Q/K163E)), xlvi)SEQ ID NO:212 (IL-12p40(E59Q/K99E)), xlvii) SEQ ID NO:213(IL-12p40(E59Q/K258E)), xlviii) SEQ ID NO:214 (IL-12p40(E59Q/K260E)),xlix) SEQ ID NO: 326 (IL-12p40 (D34N/E59K)), 1) SEQ ID NO: 325(IL-12p40(E59K/K99E)), li) SEQ ID NO: 339 (IL-12p40(D18K/E59K/K99E)),lii) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), liii) SEQ ID NO: 336(IL-12p40 (E59K/K99Y)), liv) SEQ ID NO: 335 (IL-12p40 (E59Y/K99E)), lv)SEQ ID NO: 338 (IL-12p40 (E45K/E59K/K99E)), lvi) SEQ ID NO: 340(IL-12p40 (E59K/K99E/Q144E)), lvii) SEQ ID NO: 341 (IL-12p40(E59K/K99E/Q144K)), lviii) SEQ ID NO: 342 (IL-12p40 (E59K/K99E/R159E)),lix) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), lx) SEQ ID NO: 344(IL-12p40 (D18K/E59K/K99E/K264E)), lxi) SEQ ID NO: 360 (IL-12p40(C252S)), lxii) SEQ ID NO: 361 (IL-12p40 (DI8K/E59K/K99E/C252S)), lxiii)SEQ ID NO: 362 (IL-12p40 (D18K/E59K/K99E/C252S/K264E)), lxiv) SEQ ID NO:363 (IL-12p40 (E59K/K99Y/C252S)), lxv) SEQ ID NO: 364 (IL-12p40(E59K/K99E/C252S/K264E)), lxvi) SEQ ID NO: 365 (IL-12p40(E59K/K99E/C252S)), lxvii) SEQ ID NO: 254 (IL-12p40 (N103D/N113D)),lxviii) SEQ ID NO: 255 (IL-12p40 (N103D/N200D)), lxix) SEQ ID NO: 256(IL-12p40 (N103D/N281D)), lxx) SEQ ID NO: 257 (IL-12p40 (N113D/N200D)),lxxi) SEQ ID NO: 258 (IL-12p40 (N113D/N281D)), lxxii) SEQ ID NO: 259(IL-12p40 (N200D/N281D)), lxxiii) SEQ ID NO: 260 (IL-12p40(N103D/N113D/N200D)), lxxiv) SEQ ID NO: 261 (IL-12p40(N103D/N113D/N281D)), lxxv) SEQ ID NO: 262 (IL-12p40(N103D/N200D/N281D)), lxxvi) SEQ ID NO: 263 (IL-12p40(N113D/N200D/N281D)), lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)),lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)), lxxix) SEQ ID NO: 266(IL-12p40 (N200Q)), lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)), lxxxi) SEQID NO: 268 (IL-12p40 (N103Q/N113Q)), lxxxii) SEQ ID NO: 269 (IL-12p40(N103Q/N200Q)), lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q/N281Q)),lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q/N200Q)), lxxxv) SEQ ID NO: 272(IL-12p40 (N113Q/N281Q)), lxxxvi) SEQ ID NO: 273 (IL-12p40(N200Q/N281Q)), lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q/N113Q/N200Q)),lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q/N113Q/N281Q)), lxxxix) SEQ IDNO: 276 (IL-12p40 (N103Q/N200Q/N281Q)), xc) SEQ ID NO: 277 (IL-12p40(N113Q/N200Q/N281Q)), xci) SEQ ID NO:278 (IL-12p40(N103Q/N113Q/N200Q/N281Q)), xcii) SEQ ID NO:327 (IL-12p40(D34N/E59K/K99E)), xciii) SEQ ID NO:328 (IL-12p40 (D34K/E59K/K99E)),xciv) SEQ ID NO:329 (IL-12p40 (E32Q/D34N/E59K/K99E)), xcv) SEQ ID NO:331(IL-12p40 (E32K/D34N/E59K/K99E)), xcvi) SEQ ID NO: 337 (IL-12p40(E59Y/K99Y)), xcvii) SEQ ID NO:366 (IL-12p40(E59K/K99E/N103Q/C252S/K264E)), xcviii) SEQ ID NO:367 (IL-12p40(E59K/K99E/N113Q/C252S/K264E)), xcix) SEQ ID NO:368 (IL-12p40(E59K/K99E/N200Q/C252S/K264E)), c) SEQ ID NO:369 (IL-12p40(E59K/K99E/N281Q/C252S/K264E)), ci) SEQ ID NO:370 (IL-12p40(E59K/K99E/N103Q/N113Q/C252S/K264E)), cii) SEQ ID NO:371 (IL-12p40(E59K/K99E/N103Q/N200Q/C252S/K264E)), ciii) SEQ ID NO:372 (IL-12p40(E59K/K99E/N103Q/N281Q/C252S/K264E)), civ) SEQ ID NO:373 (IL-12p40(E59K/K99E/N113Q/N200Q/C252S/K264E)), cv) SEQ ID NO:374 (IL-12p40(E59K/K99E/N113Q/N281Q/C252S/K264E)), cvi) SEQ ID NO:375 (IL-12p40(E59K/K99E/N200Q/N281Q/C252S/K264E)), cvii) SEQ ID NO:376 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E)), cviii) SEQ ID NO:377(IL-12p40 (E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E)), and cix) SEQ IDNO:378 (IL-12p40 (E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E)).

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidmodifications as amino acid residues selected from the group consistingof Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63,L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97,L124, M125, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163,D165, I171, R181, 1182, R183, V185, T186, D188, R189, V190, S192, Y193,N195, and A196.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidsubstitutions selected from the group consisting of N21D, Q35D, E38Q,D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A,L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K,E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has amino acidsubstitutions selected from the group consisting of N71D/N85D/N195D,N151D/E153Q, N151D/D165N, Q130E/N151D, N151D/K158E, E79Q/N151D,D55Q/N151D, N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q, N71Q/N195Q,N85Q/N195Q, N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, and N85D/N195D.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:113(IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115(IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D/N85D/N195D)), v) SEQID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii)SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(Q135E)),ix) SEQ ID NO:121 (IL-12p35(Q35D)), x) SEQ ID NO:122 (IL-12p35(Q146E)),xi) SEQ ID NO:123 (IL-12p35(N76D)), xii) SEQ ID NO:124(IL-12p35(E162Q)), xiii) SEQ ID NO:125 (IL-12p35(E163Q)), xiv)IL-12p35(N21D), xv) SEQ ID NO:333 (IL-12p35(D55Q)), xvi) IL-12p35(E79Q),xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx)SEQ ID NO:227 (IL-12p35(N151K)), xxi) SEQ ID NO:226 (IL-12p35(E153K)),xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO:225(IL-12p35(N151D/E153Q)), xxv) SEQ ID NO:228 (IL-12p35(N151D/D165N)),xxvi) SEQ ID NO:229 (IL-12p35(Q130E/N151D)), xxvii) SEQ ID NO:230(IL-12p35(N151D/K158E)), xxviii) SEQ ID NO:231 (IL-12p35(E79Q/N151D)),xxix) SEQ ID NO:232 (IL-12p35(D55Q/N151D)), xxx) SEQ ID NO:233(IL-12p35(N136D/N151D)), xxxi) SEQ ID NO:234 (IL-12p35(N21D/N151D)),xxxii) SEQ ID NO:235 (IL-12p35(E143Q/N151D)), xxxiii) SEQ ID NO: 345(IL-12p35(F96A)), xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)), xxxv) SEQ IDNO: 347 (IL-12p35(L89A)), xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)),xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)), xxxviii) SEQ ID NO: 350(IL-12p35(L75A)), xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)), xxxv) SEQ IDNO: 279 (IL-12p35 (N71Q)), xxxvi) SEQ ID NO: 280 (IL-12p35 (N85Q)),xxxvii) SEQ ID NO: 281 (IL-12p35 (N195Q)), xxxviii) SEQ ID NO: 282(IL-12p35 (N71Q/N85Q)), xxxix) SEQ ID NO: 283 (IL-12p35 (N71Q/N195Q)),xl) SEQ ID NO: 284 (IL-12p35 (N85Q/N195Q), xli) SEQ ID NO: 285 (IL-12p35(N71Q/N85Q/N195Q)), xlii) SEQ ID NO: 286 (IL-12p35 (N71D/N85D)), xliii)SEQ ID NO: 287 (IL-12p35 (N71D/N195D), xliv) SEQ ID NO: 288 (IL-12p35(N85D/N195D)), xlv) SEQ ID NO: 333 (IL-12p35 (D55Q)), and xlvi) SEQ IDNO: 334 (IL-12p35 (D55K)).

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said first and second monomers are XENP31289.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said first and second monomers are XENP31291.

In some embodiments, the present invention provides a homodimeric Fcfusion protein composition comprising a homodimeric Fc fusion proteinfor use in treating cancer in a subject.

In some embodiments, the present invention provides a one or morenucleic acids encoding a homodimeric Fc fusion protein.

In some embodiments, the present invention provides a host cellcomprising said one or more nucleic acids encoding a homodimeric Fcfusion protein.

In some embodiments, the present invention provides a method of making ahomodimeric Fc fusion protein, said method comprising culturing a hostcell under conditions whereby said homodimeric Fc fusion protein isproduced.

In some embodiments, the present invention provides a method ofpurifying a homodimeric Fc fusion protein, said method comprising: a)providing a composition comprising the homodimeric Fc fusion protein; b)loading said composition onto an ion exchange column; and c) collectinga fraction containing said homodimeric Fc fusion protein.

In another aspect, the present invention provides a homodimeric Fcfusion protein comprising a first monomer and a second monomer eachcomprising, from N- to C-terminal, an Fc domain-an optional first domainlinker-an IL-12p35 subunit domain-an optional second domain linker-anIL-12p40 subunit domain.

In some embodiments, the present invention provides a homodimeric Fcfusion protein, wherein said modifications promoting homodimerization ofsaid Fc domain are a set of amino acid substitutions selected from thegroup consisting of L368D/K370S; S364K; S364K/E357L; S364K/E357Q;T411E/K360E/Q362E; D401K; T366S/L368A/Y407V; T366W;T366S/L368A/Y407V/Y349C; and T366W/S354C, according to EU numbering.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said first domain linker and said second domainlinker have the same amino acid sequence.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said Fc domain has an additional set of aminoacid substitutions comprising Q295E/N384D/Q418E/N421D, according to EUnumbering.

In some embodiments, the present invention provides a homodimeric Fcfusion protein, wherein said Fc domain has an additional set of aminoacid substitutions selected from the group consisting of G236R/L328R,E233P/L234V/L235A/G236_/S239K, E233P/L234V/L235A/G236_/S239K/A327G,E233P/L234V/L235A/G236_/S267K/A327G, E233P/L234V/L235A/G236_, andE233P/L234V/L235A/G236_/S267K, according to EU numbering.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected form the group consisting of SEQ ID NO:3 (human IL-12 subunitbeta (IL-12p40) precursor sequence) and SEQ ID NO:4 (human IL-12 subunitbeta (IL-12p40) mature form sequence), and said IL-12p35 subunit has apolypeptide sequence selected from the group consisting of SEQ ID NO:1(human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ IDNO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein each of said Fc domain further comprises aminoacid substitutions M428L/N434S.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit and/or said IL-12p35 subunit is a variant IL-12p35 subunit.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex; and/or said IL-12p35 subunit is a variant IL-12p35subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidmodifications at amino acid residues selected from the group consistingof E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32,E33, D34, L40, D41, Q42, S43, E45, L47, T54, 155, Q56, K58, E59, F60,G61, D62, Q65, Y66, E73, K84, E86, D87, G88, 189, W90, D93, D97, K99,E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156,R159, D161, N162, K163, D166, D170, Q172, D174, A176, C177, P178, A179,A180, E181, S183, P185, E187, N200, S204, F206, R208, D209, D214, N218,Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F247, S248, C252S,Q256, K158, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292,Y293, and E299.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidsubstitutions selected from the group consisting of D18N, D18K, E32Q,E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q,D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N,R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N,K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has amino acidsubstitutions selected from the group consisting ofN103D/N113D/N200D/N281D, Q42E/E45Q, E45Q/Q56E, Q42E/E59Q, Q56E/E59Q,Q42E/E45Q/Q56E, E45Q/Q56E/E59Q, E32Q/E59Q, D34N/E59K, D34N/E59K/K99E,D34K/E59K/K99E, E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q,E59Q/E187Q, S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E,E59Q/K260E, E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y,E59Y/K99Y, E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:57(IL-12p40(N103D)), ii) SEQ ID NO:58 (IL-12p40(N113D)), iii) SEQ ID NO:59(IL-12p40(N200D)), iv) SEQ ID NO:60 (IL-12p40(N281D)), v) SEQ ID NO:61(IL-12p40(N103D/N113D/N200D/N281D)), vi) SEQ ID NO:62 (IL-12p40(Q42E)),vii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)),ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi)SEQ ID NO:67 (IL-12p40(Q42E/E45Q)), xii) SEQ ID NO:68(IL-12p40(E45Q/Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E/E59Q)), xiv)SEQ ID NO:70 (IL-12p40(Q56E/E59Q)), xv) SEQ ID NO:71(IL-12p40(Q42E/E45Q/Q56E)), xvi) SEQ ID NO:72(IL-12p40(E45Q/Q56E/E59Q)), xvii) SEQ ID NO:73 (IL-12p40(D161N)), xviii)SEQ ID NO:74 (IL-12p40(E73Q)), xix) SEQ ID NO:75 (IL-12p40(Q144E)), xx)SEQ ID NO:76 (IL-12p40(E262Q)), xxi) SEQ ID NO:77 (IL-12p40(E100Q)),xxii) SEQ ID NO:78 (IL-12p40(D18N)), xxiii) SEQ ID NO:79(IL-12p40(E33Q)), xxiv) SEQ ID NO:80 (IL-12p40(Q229E)), xxv) SEQ IDNO:81 (IL-12p40(E235Q)), xxvi) SEQ ID NO:82 (IL-12p40(Q256N)), xxvii)SEQ ID NO:83 (IL-12p40(E299Q)), xxviii) SEQ ID NO:84 (IL-12p40(D87N)),xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii)IL-12p40(S43K), xxxiii) SEQ ID NO:379 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E)), xxxiv) SEQ ID NO:205(IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii)IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl)SEQ ID NO:206 (IL-12p40(E32Q/E59Q)), xli) SEQ ID NO:207(IL-12p40(D34N/E59Q)), xlii) SEQ ID NO:208 (IL-12p40(E59Q/E187Q)),xliii) SEQ ID NO:209 (IL-12p40(S43E/E59Q)), xliv) SEQ ID NO:210(IL-12p40(S43K/E49Q)), xlv) SEQ ID NO:211 (IL-12p40(E59Q/K163E)), xlvi)SEQ ID NO:212 (IL-12p40(E59Q/K99E)), xlvii) SEQ ID NO:213(IL-12p40(E59Q/K258E)), xlviii) SEQ ID NO:214 (IL-12p40(E59Q/K260E)),xlix) SEQ ID NO: 326 (IL-12p40 (D34N/E59K)), 1) SEQ ID NO: 325(IL-12p40(E59K/K99E)), li) SEQ ID NO: 339 (IL-12p40(D18K/E59K/K99E)),lii) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), liii) SEQ ID NO: 336(IL-12p40 (E59K/K99Y)), liv) SEQ ID NO: 335 (IL-12p40 (E59Y/K99E)), lv)SEQ ID NO: 338 (IL-12p40 (E45K/E59K/K99E)), lvi) SEQ ID NO: 340(IL-12p40 (E59K/K99E/Q144E)), lvii) SEQ ID NO: 341 (IL-12p40(E59K/K99E/Q144K)), lviii) SEQ ID NO: 342 (IL-12p40 (E59K/K99E/R159E)),lix) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), lx) SEQ ID NO: 344(IL-12p40 (D18K/E59K/K99E/K264E)), lxi) SEQ ID NO: 360 (IL-12p40(C252S)), lxii) SEQ ID NO: 361 (IL-12p40 (DI8K/E59K/K99E/C252S)), lxiii)SEQ ID NO: 362 (IL-12p40 (D18K/E59K/K99E/C252S/K264E)), lxiv) SEQ ID NO:363 (IL-12p40 (E59K/K99Y/C252S)), lxv) SEQ ID NO: 364 (IL-12p40(E59K/K99E/C252S/K264E)), lxvi) SEQ ID NO: 365 (IL-12p40(E59K/K99E/C252S)), lxvii) SEQ ID NO: 254 (IL-12p40 (N103D/N113D)),lxviii) SEQ ID NO: 255 (IL-12p40 (N103D/N200D)), lxix) SEQ ID NO: 256(IL-12p40 (N103D/N281D)), lxx) SEQ ID NO: 257 (IL-12p40 (N113D/N200D)),lxxi) SEQ ID NO: 258 (IL-12p40 (N113D/N281D)), lxxii) SEQ ID NO: 259(IL-12p40 (N200D/N281D)), lxxiii) SEQ ID NO: 260 (IL-12p40(N103D/N113D/N200D)), lxxiv) SEQ ID NO: 261 (IL-12p40(N103D/N113D/N281D)), lxxv) SEQ ID NO: 262 (IL-12p40(N103D/N200D/N281D)), lxxvi) SEQ ID NO: 263 (IL-12p40(N113D/N200D/N281D)), lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)),lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)), lxxix) SEQ ID NO: 266(IL-12p40 (N200Q)), lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)), lxxxi) SEQID NO: 268 (IL-12p40 (N103Q/N113Q)), lxxxii) SEQ ID NO: 269 (IL-12p40(N103Q/N200Q)), lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q/N281Q)),lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q/N200Q)), lxxxv) SEQ ID NO: 272(IL-12p40 (N113Q/N281Q)), lxxxvi) SEQ ID NO: 273 (IL-12p40(N200Q/N281Q)), lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q/N113Q/N200Q)),lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q/N113Q/N281Q)), lxxxix) SEQ IDNO: 276 (IL-12p40 (N103Q/N200Q/N281Q)), xc) SEQ ID NO: 277 (IL-12p40(N113Q/N200Q/N281Q)), xci) SEQ ID NO:278 (IL-12p40(N103Q/N113Q/N200Q/N281Q)), xcii) SEQ ID NO:327 (IL-12p40(D34N/E59K/K99E)), xciii) SEQ ID NO:328 (IL-12p40 (D34K/E59K/K99E)),xciv) SEQ ID NO:329 (IL-12p40 (E32Q/D34N/E59K/K99E)), xcv) SEQ ID NO:331(IL-12p40 (E32K/D34N/E59K/K99E)), xcvi) SEQ ID NO: 337 (IL-12p40(E59Y/K99Y)), xcvii) SEQ ID NO:366 (IL-12p40(E59K/K99E/N103Q/C252S/K264E)), xcviii) SEQ ID NO:367 (IL-12p40(E59K/K99E/N113Q/C252S/K264E)), xcix) SEQ ID NO:368 (IL-12p40(E59K/K99E/N200Q/C252S/K264E)), c) SEQ ID NO:369 (IL-12p40(E59K/K99E/N281Q/C252S/K264E)), ci) SEQ ID NO:370 (IL-12p40(E59K/K99E/N103Q/N113Q/C252S/K264E)), cii) SEQ ID NO:371 (IL-12p40(E59K/K99E/N103Q/N200Q/C252S/K264E)), ciii) SEQ ID NO:372 (IL-12p40(E59K/K99E/N103Q/N281Q/C252S/K264E)), civ) SEQ ID NO:373 (IL-12p40(E59K/K99E/N113Q/N200Q/C252S/K264E)), cv) SEQ ID NO:374 (IL-12p40(E59K/K99E/N113Q/N281Q/C252S/K264E)), cvi) SEQ ID NO:375 (IL-12p40(E59K/K99E/N200Q/N281Q/C252S/K264E)), cvii) SEQ ID NO:376 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E)), cviii) SEQ ID NO:377(IL-12p40 (E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E)), and cix) SEQ IDNO:378 (IL-12p40 (E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E)).

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidmodifications as amino acid residues selected from the group consistingof Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63,L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97,L124, M125, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163,D165, I171, R181, 1182, R183, V185, T186, D188, R189, V190, S192, Y193,N195, and A196.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidsubstitutions selected from the group consisting of N21D, Q35D, E38Q,D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A,L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K,E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has amino acidsubstitutions selected from the group consisting of N71D/N85D/N195D,N151D/E153Q, N151D/D165N, Q130E/N151D, N151D/K158E, E79Q/N151D,D55Q/N151D, N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q, N71Q/N195Q,N85Q/N195Q, N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, and N85D/N195D.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:113(IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115(IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D/N85D/N195D)), v) SEQID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii)SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(Q135E)),ix) SEQ ID NO:121 (IL-12p35(Q35D)), x) SEQ ID NO:122 (IL-12p35(Q146E)),xi) SEQ ID NO:123 (IL-12p35(N76D)), xii) SEQ ID NO:124(IL-12p35(E162Q)), xiii) SEQ ID NO:125 (IL-12p35(E163Q)), xiv)IL-12p35(N21D), xv) SEQ ID NO:333 (IL-12p35(D55Q)), xvi) IL-12p35(E79Q),xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx)SEQ ID NO:227 (IL-12p35(N151K)), xxi) SEQ ID NO:226 (IL-12p35(E153K)),xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO:225(IL-12p35(N151D/E153Q)), xxv) SEQ ID NO:228 (IL-12p35(N151D/D165N)),xxvi) SEQ ID NO:229 (IL-12p35(Q130E/N151D)), xxvii) SEQ ID NO:230(IL-12p35(N151D/K158E)), xxviii) SEQ ID NO:231 (IL-12p35(E79Q/N151D)),xxix) SEQ ID NO:232 (IL-12p35(D55Q/N151D)), xxx) SEQ ID NO:233(IL-12p35(N136D/N151D)), xxxi) SEQ ID NO:234 (IL-12p35(N21D/N151D)),xxxii) SEQ ID NO:235 (IL-12p35(E143Q/N151D)), xxxiii) SEQ ID NO: 345(IL-12p35(F96A)), xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)), xxxv) SEQ IDNO: 347 (IL-12p35(L89A)), xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)),xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)), xxxviii) SEQ ID NO: 350(IL-12p35(L75A)), xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)), xxxv) SEQ IDNO: 279 (IL-12p35 (N71Q)), xxxvi) SEQ ID NO: 280 (IL-12p35 (N85Q)),xxxvii) SEQ ID NO: 281 (IL-12p35 (N195Q)), xxxviii) SEQ ID NO: 282(IL-12p35 (N71Q/N85Q)), xxxix) SEQ ID NO: 283 (IL-12p35 (N71Q/N195Q)),xl) SEQ ID NO: 284 (IL-12p35 (N85Q/N195Q), xli) SEQ ID NO: 285 (IL-12p35(N71Q/N85Q/N195Q)), xlii) SEQ ID NO: 286 (IL-12p35 (N71D/N85D)), xliii)SEQ ID NO: 287 (IL-12p35 (N71D/N195D), xliv) SEQ ID NO: 288 (IL-12p35(N85D/N195D)), xlv) SEQ ID NO: 333 (IL-12p35 (D55Q)), and xlvi) SEQ IDNO: 334 (IL-12p35 (D55K)).

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said first and second monomers are XENP31289.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said first and second monomers are XENP31291.

In some embodiments, the present invention provides a homodimeric Fcfusion protein composition comprising a homodimeric Fc fusion proteinfor use in treating cancer in a subject.

In some embodiments, the present invention provides a one or morenucleic acids encoding a homodimeric Fc fusion protein.

In some embodiments, the present invention provides a host cellcomprising said one or more nucleic acids encoding a homodimeric Fcfusion protein.

In some embodiments, the present invention provides a method of making ahomodimeric Fc fusion protein, said method comprising culturing a hostcell under conditions whereby said homodimeric Fc fusion protein isproduced.

In some embodiments, the present invention provides a method ofpurifying a homodimeric Fc fusion protein, said method comprising: a)providing a composition comprising the homodimeric Fc fusion protein; b)loading said composition onto an ion exchange column; and c) collectinga fraction containing said homodimeric Fc fusion protein.

In another aspect, the present invention provides a homodimeric Fcfusion protein comprising a first monomer and a second monomer eachcomprising, from N- to C-terminal, an IL-12p40 subunit domain-a firstdomain linker-an IL-12p35 subunit domain-a second domain linker-Fcdomain.

In some embodiments, the present invention provides a homodimeric Fcfusion protein, wherein said modifications promoting homodimerization ofsaid Fc domain are a set of amino acid substitutions selected from thegroup consisting of L368D/K370S; S364K; S364K/E357L; S364K/E357Q;T411E/K360E/Q362E; D401K; T366S/L368A/Y407V; T366W;T366S/L368A/Y407V/Y349C; and T366W/S354C, according to EU numbering.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said first domain linker and said second domainlinker have the same amino acid sequence.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said Fc domain has an additional set of aminoacid substitutions comprising Q295E/N384D/Q418E/N421D, according to EUnumbering.

In some embodiments, the present invention provides a homodimeric Fcfusion protein, wherein said Fc domain has an additional set of aminoacid substitutions selected from the group consisting of G236R/L328R,E233P/L234V/L235A/G236_/S239K, E233P/L234V/L235A/G236_/S239K/A327G,E233P/L234V/L235A/G236_/S267K/A327G, E233P/L234V/L235A/G236_, andE233P/L234V/L235A/G236_/S267K, according to EU numbering.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected form the group consisting of SEQ ID NO:3 (human IL-12 subunitbeta (IL-12p40) precursor sequence) and SEQ ID NO:4 (human IL-12 subunitbeta (IL-12p40) mature form sequence), and said IL-12p35 subunit has apolypeptide sequence selected from the group consisting of SEQ ID NO:1(human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ IDNO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein each of said Fc domain further comprises aminoacid substitutions M428L/N434S.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit and/or said IL-12p35 subunit is a variant IL-12p35 subunit.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex; and/or said IL-12p35 subunit is a variant IL-12p35subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidmodifications at amino acid residues selected from the group consistingof E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32,E33, D34, L40, D41, Q42, S43, E45, L47, T54, 155, Q56, K58, E59, F60,G61, D62, Q65, Y66, E73, K84, E86, D87, G88, 189, W90, D93, D97, K99,E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156,R159, D161, N162, K163, D166, D170, Q172, D174, A176, C177, P178, A179,A180, E181, S183, P185, E187, N200, S204, F206, R208, D209, D214, N218,Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F247, S248, C252S,Q256, K158, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292,Y293, and E299.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidsubstitutions selected from the group consisting of D18N, D18K, E32Q,E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q,D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N,R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N,K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has amino acidsubstitutions selected from the group consisting ofN103D/N113D/N200D/N281D, Q42E/E45Q, E45Q/Q56E, Q42E/E59Q, Q56E/E59Q,Q42E/E45Q/Q56E, E45Q/Q56E/E59Q, E32Q/E59Q, D34N/E59K, D34N/E59K/K99E,D34K/E59K/K99E, E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q,E59Q/E187Q, S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E,E59Q/K260E, E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y,E59Y/K99Y, E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:57(IL-12p40(N103D)), ii) SEQ ID NO:58 (IL-12p40(N113D)), iii) SEQ ID NO:59(IL-12p40(N200D)), iv) SEQ ID NO:60 (IL-12p40(N281D)), v) SEQ ID NO:61(IL-12p40(N103D/N113D/N200D/N281D)), vi) SEQ ID NO:62 (IL-12p40(Q42E)),vii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)),ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi)SEQ ID NO:67 (IL-12p40(Q42E/E45Q)), xii) SEQ ID NO:68(IL-12p40(E45Q/Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E/E59Q)), xiv)SEQ ID NO:70 (IL-12p40(Q56E/E59Q)), xv) SEQ ID NO:71(IL-12p40(Q42E/E45Q/Q56E)), xvi) SEQ ID NO:72(IL-12p40(E45Q/Q56E/E59Q)), xvii) SEQ ID NO:73 (IL-12p40(D161N)), xviii)SEQ ID NO:74 (IL-12p40(E73Q)), xix) SEQ ID NO:75 (IL-12p40(Q144E)), xx)SEQ ID NO:76 (IL-12p40(E262Q)), xxi) SEQ ID NO:77 (IL-12p40(E100Q)),xxii) SEQ ID NO:78 (IL-12p40(D18N)), xxiii) SEQ ID NO:79(IL-12p40(E33Q)), xxiv) SEQ ID NO:80 (IL-12p40(Q229E)), xxv) SEQ IDNO:81 (IL-12p40(E235Q)), xxvi) SEQ ID NO:82 (IL-12p40(Q256N)), xxvii)SEQ ID NO:83 (IL-12p40(E299Q)), xxviii) SEQ ID NO:84 (IL-12p40(D87N)),xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii)IL-12p40(S43K), xxxiii) SEQ ID NO:379 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E)), xxxiv) SEQ ID NO:205(IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii)IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl)SEQ ID NO:206 (IL-12p40(E32Q/E59Q)), xli) SEQ ID NO:207(IL-12p40(D34N/E59Q)), xlii) SEQ ID NO:208 (IL-12p40(E59Q/E187Q)),xliii) SEQ ID NO:209 (IL-12p40(S43E/E59Q)), xliv) SEQ ID NO:210(IL-12p40(S43K/E49Q)), xlv) SEQ ID NO:211 (IL-12p40(E59Q/K163E)), xlvi)SEQ ID NO:212 (IL-12p40(E59Q/K99E)), xlvii) SEQ ID NO:213(IL-12p40(E59Q/K258E)), xlviii) SEQ ID NO:214 (IL-12p40(E59Q/K260E)),xlix) SEQ ID NO: 326 (IL-12p40 (D34N/E59K)), 1) SEQ ID NO: 325(IL-12p40(E59K/K99E)), li) SEQ ID NO: 339 (IL-12p40(D18K/E59K/K99E)),lii) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), liii) SEQ ID NO: 336(IL-12p40 (E59K/K99Y)), liv) SEQ ID NO: 335 (IL-12p40 (E59Y/K99E)), lv)SEQ ID NO: 338 (IL-12p40 (E45K/E59K/K99E)), lvi) SEQ ID NO: 340(IL-12p40 (E59K/K99E/Q144E)), lvii) SEQ ID NO: 341 (IL-12p40(E59K/K99E/Q144K)), lviii) SEQ ID NO: 342 (IL-12p40 (E59K/K99E/R159E)),lix) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), lx) SEQ ID NO: 344(IL-12p40 (D18K/E59K/K99E/K264E)), lxi) SEQ ID NO: 360 (IL-12p40(C252S)), lxii) SEQ ID NO: 361 (IL-12p40 (DI8K/E59K/K99E/C252S)), lxiii)SEQ ID NO: 362 (IL-12p40 (D18K/E59K/K99E/C252S/K264E)), lxiv) SEQ ID NO:363 (IL-12p40 (E59K/K99Y/C252S)), lxv) SEQ ID NO: 364 (IL-12p40(E59K/K99E/C252S/K264E)), lxvi) SEQ ID NO: 365 (IL-12p40(E59K/K99E/C252S)), lxvii) SEQ ID NO: 254 (IL-12p40 (N103D/N113D)),lxviii) SEQ ID NO: 255 (IL-12p40 (N103D/N200D)), lxix) SEQ ID NO: 256(IL-12p40 (N103D/N281D)), lxx) SEQ ID NO: 257 (IL-12p40 (N113D/N200D)),lxxi) SEQ ID NO: 258 (IL-12p40 (N113D/N281D)), lxxii) SEQ ID NO: 259(IL-12p40 (N200D/N281D)), lxxiii) SEQ ID NO: 260 (IL-12p40(N103D/N113D/N200D)), lxxiv) SEQ ID NO: 261 (IL-12p40(N103D/N113D/N281D)), lxxv) SEQ ID NO: 262 (IL-12p40(N103D/N200D/N281D)), lxxvi) SEQ ID NO: 263 (IL-12p40(N113D/N200D/N281D)), lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)),lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)), lxxix) SEQ ID NO: 266(IL-12p40 (N200Q)), lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)), lxxxi) SEQID NO: 268 (IL-12p40 (N103Q/N113Q)), lxxxii) SEQ ID NO: 269 (IL-12p40(N103Q/N200Q)), lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q/N281Q)),lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q/N200Q)), lxxxv) SEQ ID NO: 272(IL-12p40 (N113Q/N281Q)), lxxxvi) SEQ ID NO: 273 (IL-12p40(N200Q/N281Q)), lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q/N113Q/N200Q)),lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q/N113Q/N281Q)), lxxxix) SEQ IDNO: 276 (IL-12p40 (N103Q/N200Q/N281Q)), xc) SEQ ID NO: 277 (IL-12p40(N113Q/N200Q/N281Q)), xci) SEQ ID NO:278 (IL-12p40(N103Q/N113Q/N200Q/N281Q)), xcii) SEQ ID NO:327 (IL-12p40(D34N/E59K/K99E)), xciii) SEQ ID NO:328 (IL-12p40 (D34K/E59K/K99E)),xciv) SEQ ID NO:329 (IL-12p40 (E32Q/D34N/E59K/K99E)), xcv) SEQ ID NO:331(IL-12p40 (E32K/D34N/E59K/K99E)), xcvi) SEQ ID NO: 337 (IL-12p40(E59Y/K99Y)), xcvii) SEQ ID NO:366 (IL-12p40(E59K/K99E/N103Q/C252S/K264E)), xcviii) SEQ ID NO:367 (IL-12p40(E59K/K99E/N113Q/C252S/K264E)), xcix) SEQ ID NO:368 (IL-12p40(E59K/K99E/N200Q/C252S/K264E)), c) SEQ ID NO:369 (IL-12p40(E59K/K99E/N281Q/C252S/K264E)), ci) SEQ ID NO:370 (IL-12p40(E59K/K99E/N103Q/N113Q/C252S/K264E)), cii) SEQ ID NO:371 (IL-12p40(E59K/K99E/N103Q/N200Q/C252S/K264E)), ciii) SEQ ID NO:372 (IL-12p40(E59K/K99E/N103Q/N281Q/C252S/K264E)), civ) SEQ ID NO:373 (IL-12p40(E59K/K99E/N113Q/N200Q/C252S/K264E)), cv) SEQ ID NO:374 (IL-12p40(E59K/K99E/N113Q/N281Q/C252S/K264E)), cvi) SEQ ID NO:375 (IL-12p40(E59K/K99E/N200Q/N281Q/C252S/K264E)), cvii) SEQ ID NO:376 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E)), cviii) SEQ ID NO:377(IL-12p40 (E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E)), and cix) SEQ IDNO:378 (IL-12p40 (E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E)).

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidmodifications as amino acid residues selected from the group consistingof Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63,L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97,L124, M125, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163,D165, I171, R181, 1182, R183, V185, T186, D188, R189, V190, S192, Y193,N195, and A196.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidsubstitutions selected from the group consisting of N21D, Q35D, E38Q,D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A,L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K,E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has amino acidsubstitutions selected from the group consisting of N71D/N85D/N195D,N151D/E153Q, N151D/D165N, Q130E/N151D, N151D/K158E, E79Q/N151D,D55Q/N151D, N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q, N71Q/N195Q,N85Q/N195Q, N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, and N85D/N195D.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:113(IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115(IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D/N85D/N195D)), v) SEQID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii)SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(Q135E)),ix) SEQ ID NO:121 (IL-12p35(Q35D)), x) SEQ ID NO:122 (IL-12p35(Q146E)),xi) SEQ ID NO:123 (IL-12p35(N76D)), xii) SEQ ID NO:124(IL-12p35(E162Q)), xiii) SEQ ID NO:125 (IL-12p35(E163Q)), xiv)IL-12p35(N21D), xv) SEQ ID NO:333 (IL-12p35(D55Q)), xvi) IL-12p35(E79Q),xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx)SEQ ID NO:227 (IL-12p35(N151K)), xxi) SEQ ID NO:226 (IL-12p35(E153K)),xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO:225(IL-12p35(N151D/E153Q)), xxv) SEQ ID NO:228 (IL-12p35(N151D/D165N)),xxvi) SEQ ID NO:229 (IL-12p35(Q130E/N151D)), xxvii) SEQ ID NO:230(IL-12p35(N151D/K158E)), xxviii) SEQ ID NO:231 (IL-12p35(E79Q/N151D)),xxix) SEQ ID NO:232 (IL-12p35(D55Q/N151D)), xxx) SEQ ID NO:233(IL-12p35(N136D/N151D)), xxxi) SEQ ID NO:234 (IL-12p35(N21D/N151D)),xxxii) SEQ ID NO:235 (IL-12p35(E143Q/N151D)), xxxiii) SEQ ID NO: 345(IL-12p35(F96A)), xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)), xxxv) SEQ IDNO: 347 (IL-12p35(L89A)), xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)),xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)), xxxviii) SEQ ID NO: 350(IL-12p35(L75A)), xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)), xxxv) SEQ IDNO: 279 (IL-12p35 (N71Q)), xxxvi) SEQ ID NO: 280 (IL-12p35 (N85Q)),xxxvii) SEQ ID NO: 281 (IL-12p35 (N195Q)), xxxviii) SEQ ID NO: 282(IL-12p35 (N71Q/N85Q)), xxxix) SEQ ID NO: 283 (IL-12p35 (N71Q/N195Q)),xl) SEQ ID NO: 284 (IL-12p35 (N85Q/N195Q), xli) SEQ ID NO: 285 (IL-12p35(N71Q/N85Q/N195Q)), xlii) SEQ ID NO: 286 (IL-12p35 (N71D/N85D)), xliii)SEQ ID NO: 287 (IL-12p35 (N71D/N195D), xliv) SEQ ID NO: 288 (IL-12p35(N85D/N195D)), xlv) SEQ ID NO: 333 (IL-12p35 (D55Q)), and xlvi) SEQ IDNO: 334 (IL-12p35 (D55K)).

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said first and second monomers are XENP31289.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said first and second monomers are XENP31291.

In some embodiments, the present invention provides a homodimeric Fcfusion protein composition comprising a homodimeric Fc fusion proteinfor use in treating cancer in a subject.

In some embodiments, the present invention provides a one or morenucleic acids encoding a homodimeric Fc fusion protein.

In some embodiments, the present invention provides a host cellcomprising said one or more nucleic acids encoding a homodimeric Fcfusion protein.

In some embodiments, the present invention provides a method of making ahomodimeric Fc fusion protein, said method comprising culturing a hostcell under conditions whereby said homodimeric Fc fusion protein isproduced.

In some embodiments, the present invention provides a method ofpurifying a homodimeric Fc fusion protein, said method comprising: a)providing a composition comprising the homodimeric Fc fusion protein; b)loading said composition onto an ion exchange column; and c) collectinga fraction containing said homodimeric Fc fusion protein.

In another aspect, the present invention provides a homodimeric Fcfusion protein comprising a first monomer and a second monomer eachcomprising, from N- to C-terminal, an IL-12p35 subunit domain-a firstdomain linker-an IL-12p40 subunit domain-a second domain linker-Fcdomain.

In some embodiments, the present invention provides a homodimeric Fcfusion protein, wherein said modifications promoting homodimerization ofsaid Fc domain are a set of amino acid substitutions selected from thegroup consisting of L368D/K370S; S364K; S364K/E357L; S364K/E357Q;T411E/K360E/Q362E; D401K; T366S/L368A/Y407V; T366W;T366S/L368A/Y407V/Y349C; and T366W/S354C, according to EU numbering.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said first domain linker and said second domainlinker have the same amino acid sequence.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said Fc domain has an additional set of aminoacid substitutions comprising Q295E/N384D/Q418E/N421D, according to EUnumbering.

In some embodiments, the present invention provides a homodimeric Fcfusion protein, wherein said Fc domain has an additional set of aminoacid substitutions selected from the group consisting of G236R/L328R,E233P/L234V/L235A/G236_/S239K, E233P/L234V/L235A/G236_/S239K/A327G,E233P/L234V/L235A/G236_/S267K/A327G, E233P/L234V/L235A/G236_, andE233P/L234V/L235A/G236_/S267K, according to EU numbering.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected form the group consisting of SEQ ID NO:3 (human IL-12 subunitbeta (IL-12p40) precursor sequence) and SEQ ID NO:4 (human IL-12 subunitbeta (IL-12p40) mature form sequence), and said IL-12p35 subunit has apolypeptide sequence selected from the group consisting of SEQ ID NO:1(human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ IDNO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein each of said Fc domain further comprises aminoacid substitutions M428L/N434S.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit and/or said IL-12p35 subunit is a variant IL-12p35 subunit.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex; and/or said IL-12p35 subunit is a variant IL-12p35subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidmodifications at amino acid residues selected from the group consistingof E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32,E33, D34, L40, D41, Q42, S43, E45, L47, T54, 155, Q56, K58, E59, F60,G61, D62, Q65, Y66, E73, K84, E86, D87, G88, 189, W90, D93, D97, K99,E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156,R159, D161, N162, K163, D166, D170, Q172, D174, A176, C177, P178, A179,A180, E181, S183, P185, E187, N200, S204, F206, R208, D209, D214, N218,Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F247, S248, C252S,Q256, K158, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292,Y293, and E299.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidsubstitutions selected from the group consisting of D18N, D18K, E32Q,E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q,D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N,R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N,K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has amino acidsubstitutions selected from the group consisting ofN103D/N113D/N200D/N281D, Q42E/E45Q, E45Q/Q56E, Q42E/E59Q, Q56E/E59Q,Q42E/E45Q/Q56E, E45Q/Q56E/E59Q, E32Q/E59Q, D34N/E59K, D34N/E59K/K99E,D34K/E59K/K99E, E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q,E59Q/E187Q, S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E,E59Q/K260E, E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y,E59Y/K99Y, E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:57(IL-12p40(N103D)), ii) SEQ ID NO:58 (IL-12p40(N113D)), iii) SEQ ID NO:59(IL-12p40(N200D)), iv) SEQ ID NO:60 (IL-12p40(N281D)), v) SEQ ID NO:61(IL-12p40(N103D/N113D/N200D/N281D)), vi) SEQ ID NO:62 (IL-12p40(Q42E)),vii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)),ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi)SEQ ID NO:67 (IL-12p40(Q42E/E45Q)), xii) SEQ ID NO:68(IL-12p40(E45Q/Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E/E59Q)), xiv)SEQ ID NO:70 (IL-12p40(Q56E/E59Q)), xv) SEQ ID NO:71(IL-12p40(Q42E/E45Q/Q56E)), xvi) SEQ ID NO:72(IL-12p40(E45Q/Q56E/E59Q)), xvii) SEQ ID NO:73 (IL-12p40(D161N)), xviii)SEQ ID NO:74 (IL-12p40(E73Q)), xix) SEQ ID NO:75 (IL-12p40(Q144E)), xx)SEQ ID NO:76 (IL-12p40(E262Q)), xxi) SEQ ID NO:77 (IL-12p40(E100Q)),xxii) SEQ ID NO:78 (IL-12p40(D18N)), xxiii) SEQ ID NO:79(IL-12p40(E33Q)), xxiv) SEQ ID NO:80 (IL-12p40(Q229E)), xxv) SEQ IDNO:81 (IL-12p40(E235Q)), xxvi) SEQ ID NO:82 (IL-12p40(Q256N)), xxvii)SEQ ID NO:83 (IL-12p40(E299Q)), xxviii) SEQ ID NO:84 (IL-12p40(D87N)),xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii)IL-12p40(S43K), xxxiii) SEQ ID NO:379 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E)), xxxiv) SEQ ID NO:205(IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii)IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl)SEQ ID NO:206 (IL-12p40(E32Q/E59Q)), xli) SEQ ID NO:207(IL-12p40(D34N/E59Q)), xlii) SEQ ID NO:208 (IL-12p40(E59Q/E187Q)),xliii) SEQ ID NO:209 (IL-12p40(S43E/E59Q)), xliv) SEQ ID NO:210(IL-12p40(S43K/E49Q)), xlv) SEQ ID NO:211 (IL-12p40(E59Q/K163E)), xlvi)SEQ ID NO:212 (IL-12p40(E59Q/K99E)), xlvii) SEQ ID NO:213(IL-12p40(E59Q/K258E)), xlviii) SEQ ID NO:214 (IL-12p40(E59Q/K260E)),xlix) SEQ ID NO: 326 (IL-12p40 (D34N/E59K)), 1) SEQ ID NO: 325(IL-12p40(E59K/K99E)), li) SEQ ID NO: 339 (IL-12p40(D18K/E59K/K99E)),lii) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), liii) SEQ ID NO: 336(IL-12p40 (E59K/K99Y)), liv) SEQ ID NO: 335 (IL-12p40 (E59Y/K99E)), lv)SEQ ID NO: 338 (IL-12p40 (E45K/E59K/K99E)), lvi) SEQ ID NO: 340(IL-12p40 (E59K/K99E/Q144E)), lvii) SEQ ID NO: 341 (IL-12p40(E59K/K99E/Q144K)), lviii) SEQ ID NO: 342 (IL-12p40 (E59K/K99E/R159E)),lix) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), lx) SEQ ID NO: 344(IL-12p40 (D18K/E59K/K99E/K264E)), lxi) SEQ ID NO: 360 (IL-12p40(C252S)), lxii) SEQ ID NO: 361 (IL-12p40 (DI8K/E59K/K99E/C252S)), lxiii)SEQ ID NO: 362 (IL-12p40 (D18K/E59K/K99E/C252S/K264E)), lxiv) SEQ ID NO:363 (IL-12p40 (E59K/K99Y/C252S)), lxv) SEQ ID NO: 364 (IL-12p40(E59K/K99E/C252S/K264E)), lxvi) SEQ ID NO: 365 (IL-12p40(E59K/K99E/C252S)), lxvii) SEQ ID NO: 254 (IL-12p40 (N103D/N113D)),lxviii) SEQ ID NO: 255 (IL-12p40 (N103D/N200D)), lxix) SEQ ID NO: 256(IL-12p40 (N103D/N281D)), lxx) SEQ ID NO: 257 (IL-12p40 (N113D/N200D)),lxxi) SEQ ID NO: 258 (IL-12p40 (N113D/N281D)), lxxii) SEQ ID NO: 259(IL-12p40 (N200D/N281D)), lxxiii) SEQ ID NO: 260 (IL-12p40(N103D/N113D/N200D)), lxxiv) SEQ ID NO: 261 (IL-12p40(N103D/N113D/N281D)), lxxv) SEQ ID NO: 262 (IL-12p40(N103D/N200D/N281D)), lxxvi) SEQ ID NO: 263 (IL-12p40(N113D/N200D/N281D)), lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)),lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)), lxxix) SEQ ID NO: 266(IL-12p40 (N200Q)), lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)), lxxxi) SEQID NO: 268 (IL-12p40 (N103Q/N113Q)), lxxxii) SEQ ID NO: 269 (IL-12p40(N103Q/N200Q)), lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q/N281Q)),lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q/N200Q)), lxxxv) SEQ ID NO: 272(IL-12p40 (N113Q/N281Q)), lxxxvi) SEQ ID NO: 273 (IL-12p40(N200Q/N281Q)), lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q/N113Q/N200Q)),lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q/N113Q/N281Q)), lxxxix) SEQ IDNO: 276 (IL-12p40 (N103Q/N200Q/N281Q)), xc) SEQ ID NO: 277 (IL-12p40(N113Q/N200Q/N281Q)), xci) SEQ ID NO:278 (IL-12p40(N103Q/N113Q/N200Q/N281Q)), xcii) SEQ ID NO:327 (IL-12p40(D34N/E59K/K99E)), xciii) SEQ ID NO:328 (IL-12p40 (D34K/E59K/K99E)),xciv) SEQ ID NO:329 (IL-12p40 (E32Q/D34N/E59K/K99E)), xcv) SEQ ID NO:331(IL-12p40 (E32K/D34N/E59K/K99E)), xcvi) SEQ ID NO: 337 (IL-12p40(E59Y/K99Y)), xcvii) SEQ ID NO:366 (IL-12p40(E59K/K99E/N103Q/C252S/K264E)), xcviii) SEQ ID NO:367 (IL-12p40(E59K/K99E/N113Q/C252S/K264E)), xcix) SEQ ID NO:368 (IL-12p40(E59K/K99E/N200Q/C252S/K264E)), c) SEQ ID NO:369 (IL-12p40(E59K/K99E/N281Q/C252S/K264E)), ci) SEQ ID NO:370 (IL-12p40(E59K/K99E/N103Q/N113Q/C252S/K264E)), cii) SEQ ID NO:371 (IL-12p40(E59K/K99E/N103Q/N200Q/C252S/K264E)), ciii) SEQ ID NO:372 (IL-12p40(E59K/K99E/N103Q/N281Q/C252S/K264E)), civ) SEQ ID NO:373 (IL-12p40(E59K/K99E/N113Q/N200Q/C252S/K264E)), cv) SEQ ID NO:374 (IL-12p40(E59K/K99E/N113Q/N281Q/C252S/K264E)), cvi) SEQ ID NO:375 (IL-12p40(E59K/K99E/N200Q/N281Q/C252S/K264E)), cvii) SEQ ID NO:376 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E)), cviii) SEQ ID NO:377(IL-12p40 (E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E)), and cix) SEQ IDNO:378 (IL-12p40 (E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E)).

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidmodifications as amino acid residues selected from the group consistingof Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63,L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97,L124, M125, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163,D165, I171, R181, 1182, R183, V185, T186, D188, R189, V190, S192, Y193,N195, and A196.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidsubstitutions selected from the group consisting of N21D, Q35D, E38Q,D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A,L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K,E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has amino acidsubstitutions selected from the group consisting of N71D/N85D/N195D,N151D/E153Q, N151D/D165N, Q130E/N151D, N151D/K158E, E79Q/N151D,D55Q/N151D, N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q, N71Q/N195Q,N85Q/N195Q, N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, and N85D/N195D.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:113(IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115(IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D/N85D/N195D)), v) SEQID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii)SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(Q135E)),ix) SEQ ID NO:121 (IL-12p35(Q35D)), x) SEQ ID NO:122 (IL-12p35(Q146E)),xi) SEQ ID NO:123 (IL-12p35(N76D)), xii) SEQ ID NO:124(IL-12p35(E162Q)), xiii) SEQ ID NO:125 (IL-12p35(E163Q)), xiv)IL-12p35(N21D), xv) SEQ ID NO:333 (IL-12p35(D55Q)), xvi) IL-12p35(E79Q),xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) (IL-12p35(E143Q)),xx) SEQ ID NO:227 (IL-12p35(N151K)), xxi) SEQ ID NO:226(IL-12p35(E153K)), xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv)SEQ ID NO:225 (IL-12p35(N151D/E153Q)), xxv) SEQ ID NO:228(IL-12p35(N151D/D165N)), xxvi) SEQ ID NO:229 (IL-12p35(Q130E/N151D)),xxvii) SEQ ID NO:230 (IL-12p35(N151D/K158E)), xxviii) SEQ ID NO:231(IL-12p35(E79Q/N151D)), xxix) SEQ ID NO:232 (IL-12p35(D55Q/N151D)), xxx)SEQ ID NO:233 (IL-12p35(N136D/N151D)), xxxi) SEQ ID NO:234(IL-12p35(N21D/N151D)), xxxii) SEQ ID NO:235 (IL-12p35(E143Q/N151D)),xxxiii) SEQ ID NO: 345 (IL-12p35(F96A)), xxxiv) SEQ ID NO: 346(IL-12p35(M97A)), xxxv) SEQ ID NO: 347 (IL-12p35(L89A)), xxxvi) SEQ IDNO: 348 (IL-12p35(L124A)), xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)),xxxviii) SEQ ID NO: 350 (IL-12p35(L75A)), xxxiv) SEQ ID NO: 351(IL-12p35(I171A)), xxxv) SEQ ID NO: 279 (IL-12p35 (N71Q)), xxxvi) SEQ IDNO: 280 (IL-12p35 (N85Q)), xxxvii) SEQ ID NO: 281 (IL-12p35 (N195Q)),xxxviii) SEQ ID NO: 282 (IL-12p35 (N71Q/N85Q)), xxxix) SEQ ID NO: 283(IL-12p35 (N71Q/N195Q)), xl) SEQ ID NO: 284 (IL-12p35 (N85Q/N195Q), xli)SEQ ID NO: 285 (IL-12p35 (N71Q/N85Q/N195Q)), xlii) SEQ ID NO: 286(IL-12p35 (N71D/N85D)), xliii) SEQ ID NO: 287 (IL-12p35 (N71D/N195D),xliv) SEQ ID NO: 288 (IL-12p35 (N85D/N195D)), xlv) SEQ ID NO: 333(IL-12p35 (D55Q)), and xlvi) SEQ ID NO: 334 (IL-12p35 (D55K)).

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said first and second monomers are XENP31289.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said first and second monomers are XENP31291.

In some embodiments, the present invention provides a homodimeric Fcfusion protein composition comprising a homodimeric Fc fusion proteinfor use in treating cancer in a subject.

In some embodiments, the present invention provides a one or morenucleic acids encoding a homodimeric Fc fusion protein.

In some embodiments, the present invention provides a host cellcomprising said one or more nucleic acids encoding a homodimeric Fcfusion protein.

In some embodiments, the present invention provides a method of making ahomodimeric Fc fusion protein, said method comprising culturing a hostcell under conditions whereby said homodimeric Fc fusion protein isproduced.

In some embodiments, the present invention provides a method ofpurifying a homodimeric Fc fusion protein, said method comprising: a)providing a composition comprising the homodimeric Fc fusion protein; b)loading said composition onto an ion exchange column; and c) collectinga fraction containing said homodimeric Fc fusion protein.

In another aspect, the present invention provides a homodimeric Fcfusion protein comprising a first monomer and a second monomer eachcomprising, from N- to C-terminal, a Fc domain-a first domain linker-anIL-12p40 subunit domain-a second domain linker-an IL-12p35 subunitdomain.

In some embodiments, the present invention provides a homodimeric Fcfusion protein, wherein said modifications promoting homodimerization ofsaid Fc domain are a set of amino acid substitutions selected from thegroup consisting of L368D/K370S; S364K; S364K/E357L; S364K/E357Q;T411E/K360E/Q362E; D401K; T366S/L368A/Y407V; T366W;T366S/L368A/Y407V/Y349C; and T366W/S354C, according to EU numbering.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said first domain linker and said second domainlinker have the same amino acid sequence.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said Fc domain has an additional set of aminoacid substitutions comprising Q295E/N384D/Q418E/N421D, according to EUnumbering.

In some embodiments, the present invention provides a homodimeric Fcfusion protein, wherein said Fc domain has an additional set of aminoacid substitutions selected from the group consisting of G236R/L328R,E233P/L234V/L235A/G236_/S239K, E233P/L234V/L235A/G236_/S239K/A327G,E233P/L234V/L235A/G236_/S267K/A327G, E233P/L234V/L235A/G236_, andE233P/L234V/L235A/G236_/S267K, according to EU numbering.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected form the group consisting of SEQ ID NO:3 (human IL-12 subunitbeta (IL-12p40) precursor sequence) and SEQ ID NO:4 (human IL-12 subunitbeta (IL-12p40) mature form sequence), and said IL-12p35 subunit has apolypeptide sequence selected from the group consisting of SEQ ID NO:1(human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ IDNO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein each of said Fc domain further comprises aminoacid substitutions M428L/N434S.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit and/or said IL-12p35 subunit is a variant IL-12p35 subunit.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex; and/or said IL-12p35 subunit is a variant IL-12p35subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidmodifications at amino acid residues selected from the group consistingof E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32,E33, D34, L40, D41, Q42, S43, E45, L47, T54, 155, Q56, K58, E59, F60,G61, D62, Q65, Y66, E73, K84, E86, D87, G88, 189, W90, D93, D97, K99,E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156,R159, D161, N162, K163, D166, D170, Q172, D174, A176, C177, P178, A179,A180, E181, S183, P185, E187, N200, S204, F206, R208, D209, D214, N218,Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F247, S248, C252S,Q256, K158, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292,Y293, and E299.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidsubstitutions selected from the group consisting of D18N, D18K, E32Q,E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q,D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N,R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N,K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has amino acidsubstitutions selected from the group consisting ofN103D/N113D/N200D/N281D, Q42E/E45Q, E45Q/Q56E, Q42E/E59Q, Q56E/E59Q,Q42E/E45Q/Q56E, E45Q/Q56E/E59Q, E32Q/E59Q, D34N/E59K, D34N/E59K/K99E,D34K/E59K/K99E, E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q,E59Q/E187Q, S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E,E59Q/K260E, E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y,E59Y/K99Y, E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:57(IL-12p40(N103D)), ii) SEQ ID NO:58 (IL-12p40(N113D)), iii) SEQ ID NO:59(IL-12p40(N200D)), iv) SEQ ID NO:60 (IL-12p40(N281D)), v) SEQ ID NO:61(IL-12p40(N103D/N113D/N200D/N281D)), vi) SEQ ID NO:62 (IL-12p40(Q42E)),vii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)),ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi)SEQ ID NO:67 (IL-12p40(Q42E/E45Q)), xii) SEQ ID NO:68(IL-12p40(E45Q/Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E/E59Q)), xiv)SEQ ID NO:70 (IL-12p40(Q56E/E59Q)), xv) SEQ ID NO:71(IL-12p40(Q42E/E45Q/Q56E)), xvi) SEQ ID NO:72(IL-12p40(E45Q/Q56E/E59Q)), xvii) SEQ ID NO:73 (IL-12p40(D161N)), xviii)SEQ ID NO:74 (IL-12p40(E73Q)), xix) SEQ ID NO:75 (IL-12p40(Q144E)), xx)SEQ ID NO:76 (IL-12p40(E262Q)), xxi) SEQ ID NO:77 (IL-12p40(E100Q)),xxii) SEQ ID NO:78 (IL-12p40(D18N)), xxiii) SEQ ID NO:79(IL-12p40(E33Q)), xxiv) SEQ ID NO:80 (IL-12p40(Q229E)), xxv) SEQ IDNO:81 (IL-12p40(E235Q)), xxvi) SEQ ID NO:82 (IL-12p40(Q256N)), xxvii)SEQ ID NO:83 (IL-12p40(E299Q)), xxviii) SEQ ID NO:84 (IL-12p40(D87N)),xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii)IL-12p40(S43K), xxxiii) SEQ ID NO:379 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E)), xxxiv) SEQ ID NO:205(IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii)IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl)SEQ ID NO:206 (IL-12p40(E32Q/E59Q)), xli) SEQ ID NO:207(IL-12p40(D34N/E59Q)), xlii) SEQ ID NO:208 (IL-12p40(E59Q/E187Q)),xliii) SEQ ID NO:209 (IL-12p40(S43E/E59Q)), xliv) SEQ ID NO:210(IL-12p40(S43K/E49Q)), xlv) SEQ ID NO:211 (IL-12p40(E59Q/K163E)), xlvi)SEQ ID NO:212 (IL-12p40(E59Q/K99E)), xlvii) SEQ ID NO:213(IL-12p40(E59Q/K258E)), xlviii) SEQ ID NO:214 (IL-12p40(E59Q/K260E)),xlix) SEQ ID NO: 326 (IL-12p40 (D34N/E59K)), 1) SEQ ID NO: 325(IL-12p40(E59K/K99E)), li) SEQ ID NO: 339 (IL-12p40(D18K/E59K/K99E)),lii) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), liii) SEQ ID NO: 336(IL-12p40 (E59K/K99Y)), liv) SEQ ID NO: 335 (IL-12p40 (E59Y/K99E)), lv)SEQ ID NO: 338 (IL-12p40 (E45K/E59K/K99E)), lvi) SEQ ID NO: 340(IL-12p40 (E59K/K99E/Q144E)), lvii) SEQ ID NO: 341 (IL-12p40(E59K/K99E/Q144K)), lviii) SEQ ID NO: 342 (IL-12p40 (E59K/K99E/R159E)),lix) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), lx) SEQ ID NO: 344(IL-12p40 (D18K/E59K/K99E/K264E)), lxi) SEQ ID NO: 360 (IL-12p40(C252S)), lxii) SEQ ID NO: 361 (IL-12p40 (DI8K/E59K/K99E/C252S)), lxiii)SEQ ID NO: 362 (IL-12p40 (D18K/E59K/K99E/C252S/K264E)), lxiv) SEQ ID NO:363 (IL-12p40 (E59K/K99Y/C252S)), lxv) SEQ ID NO: 364 (IL-12p40(E59K/K99E/C252S/K264E)), lxvi) SEQ ID NO: 365 (IL-12p40(E59K/K99E/C252S)), lxvii) SEQ ID NO: 254 (IL-12p40 (N103D/N113D)),lxviii) SEQ ID NO: 255 (IL-12p40 (N103D/N200D)), lxix) SEQ ID NO: 256(IL-12p40 (N103D/N281D)), lxx) SEQ ID NO: 257 (IL-12p40 (N113D/N200D)),lxxi) SEQ ID NO: 258 (IL-12p40 (N113D/N281D)), lxxii) SEQ ID NO: 259(IL-12p40 (N200D/N281D)), lxxiii) SEQ ID NO: 260 (IL-12p40(N103D/N113D/N200D)), lxxiv) SEQ ID NO: 261 (IL-12p40(N103D/N113D/N281D)), lxxv) SEQ ID NO: 262 (IL-12p40(N103D/N200D/N281D)), lxxvi) SEQ ID NO: 263 (IL-12p40(N113D/N200D/N281D)), lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)),lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)), lxxix) SEQ ID NO: 266(IL-12p40 (N200Q)), lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)), lxxxi) SEQID NO: 268 (IL-12p40 (N103Q/N113Q)), lxxxii) SEQ ID NO: 269 (IL-12p40(N103Q/N200Q)), lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q/N281Q)),lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q/N200Q)), lxxxv) SEQ ID NO: 272(IL-12p40 (N113Q/N281Q)), lxxxvi) SEQ ID NO: 273 (IL-12p40(N200Q/N281Q)), lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q/N113Q/N200Q)),lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q/N113Q/N281Q)), lxxxix) SEQ IDNO: 276 (IL-12p40 (N103Q/N200Q/N281Q)), xc) SEQ ID NO: 277 (IL-12p40(N113Q/N200Q/N281Q)), xci) SEQ ID NO:278 (IL-12p40(N103Q/N113Q/N200Q/N281Q)), xcii) SEQ ID NO:327 (IL-12p40(D34N/E59K/K99E)), xciii) SEQ ID NO:328 (IL-12p40 (D34K/E59K/K99E)),xciv) SEQ ID NO:329 (IL-12p40 (E32Q/D34N/E59K/K99E)), xcv) SEQ ID NO:331(IL-12p40 (E32K/D34N/E59K/K99E)), xcvi) SEQ ID NO: 337 (IL-12p40(E59Y/K99Y)), xcvii) SEQ ID NO:366 (IL-12p40(E59K/K99E/N103Q/C252S/K264E)), xcviii) SEQ ID NO:367 (IL-12p40(E59K/K99E/N113Q/C252S/K264E)), xcix) SEQ ID NO:368 (IL-12p40(E59K/K99E/N200Q/C252S/K264E)), c) SEQ ID NO:369 (IL-12p40(E59K/K99E/N281Q/C252S/K264E)), ci) SEQ ID NO:370 (IL-12p40(E59K/K99E/N103Q/N113Q/C252S/K264E)), cii) SEQ ID NO:371 (IL-12p40(E59K/K99E/N103Q/N200Q/C252S/K264E)), ciii) SEQ ID NO:372 (IL-12p40(E59K/K99E/N103Q/N281Q/C252S/K264E)), civ) SEQ ID NO:373 (IL-12p40(E59K/K99E/N113Q/N200Q/C252S/K264E)), cv) SEQ ID NO:374 (IL-12p40(E59K/K99E/N113Q/N281Q/C252S/K264E)), cvi) SEQ ID NO:375 (IL-12p40(E59K/K99E/N200Q/N281Q/C252S/K264E)), cvii) SEQ ID NO:376 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E)), cviii) SEQ ID NO:377(IL-12p40 (E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E)), and cix) SEQ IDNO:378 (IL-12p40 (E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E)).

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidmodifications as amino acid residues selected from the group consistingof Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63,L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97,L124, M125, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163,D165, I171, R181, 1182, R183, V185, T186, D188, R189, V190, S192, Y193,N195, and A196.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidsubstitutions selected from the group consisting of N21D, Q35D, E38Q,D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A,L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K,E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has amino acidsubstitutions selected from the group consisting of N71D/N85D/N195D,N151D/E153Q, N151D/D165N, Q130E/N151D, N151D/K158E, E79Q/N151D,D55Q/N151D, N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q, N71Q/N195Q,N85Q/N195Q, N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, and N85D/N195D.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:113(IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115(IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D/N85D/N195D)), v) SEQID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii)SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(Q135E)),ix) SEQ ID NO:121 (IL-12p35(Q35D)), x) SEQ ID NO:122 (IL-12p35(Q146E)),xi) SEQ ID NO:123 (IL-12p35(N76D)), xii) SEQ ID NO:124(IL-12p35(E162Q)), xiii) SEQ ID NO:125 (IL-12p35(E163Q)), xiv)IL-12p35(N21D), xv) SEQ ID NO:333 (IL-12p35(D55Q)), xvi) IL-12p35(E79Q),xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx)SEQ ID NO:227 (IL-12p35(N151K)), xxi) SEQ ID NO:226 (IL-12p35(E153K)),xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO:225(IL-12p35(N151D/E153Q)), xxv) SEQ ID NO:228 (IL-12p35(N151D/D165N)),xxvi) SEQ ID NO:229 (IL-12p35(Q130E/N151D)), xxvii) SEQ ID NO:230(IL-12p35(N151D/K158E)), xxviii) SEQ ID NO:231 (IL-12p35(E79Q/N151D)),xxix) SEQ ID NO:232 (IL-12p35(D55Q/N151D)), xxx) SEQ ID NO:233(IL-12p35(N136D/N151D)), xxxi) SEQ ID NO:234 (IL-12p35(N21D/N151D)),xxxii) SEQ ID NO:235 (IL-12p35(E143Q/N151D)), xxxiii) SEQ ID NO: 345(IL-12p35(F96A)), xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)), xxxv) SEQ IDNO: 347 (IL-12p35(L89A)), xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)),xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)), xxxviii) SEQ ID NO: 350(IL-12p35(L75A)), xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)), xxxv) SEQ IDNO: 279 (IL-12p35 (N71Q)), xxxvi) SEQ ID NO: 280 (IL-12p35 (N85Q)),xxxvii) SEQ ID NO: 281 (IL-12p35 (N195Q)), xxxviii) SEQ ID NO: 282(IL-12p35 (N71Q/N85Q)), xxxix) SEQ ID NO: 283 (IL-12p35 (N71Q/N195Q)),xl) SEQ ID NO: 284 (IL-12p35 (N85Q/N195Q), xli) SEQ ID NO: 285 (IL-12p35(N71Q/N85Q/N195Q)), xlii) SEQ ID NO: 286 (IL-12p35 (N71D/N85D)), xliii)SEQ ID NO: 287 (IL-12p35 (N71D/N195D), xliv) SEQ ID NO: 288 (IL-12p35(N85D/N195D)), xlv) SEQ ID NO: 333 (IL-12p35 (D55Q)), and xlvi) SEQ IDNO: 334 (IL-12p35 (D55K)).

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said first and second monomers are XENP31289.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said first and second monomers are XENP31291.

In some embodiments, the present invention provides a homodimeric Fcfusion protein composition comprising a homodimeric Fc fusion proteinfor use in treating cancer in a subject.

In some embodiments, the present invention provides a one or morenucleic acids encoding a homodimeric Fc fusion protein.

In some embodiments, the present invention provides a host cellcomprising said one or more nucleic acids encoding a homodimeric Fcfusion protein.

In some embodiments, the present invention provides a method of making ahomodimeric Fc fusion protein, said method comprising culturing a hostcell under conditions whereby said homodimeric Fc fusion protein isproduced.

In some embodiments, the present invention provides a method ofpurifying a homodimeric Fc fusion protein, said method comprising: a)providing a composition comprising the homodimeric Fc fusion protein; b)loading said composition onto an ion exchange column; and c) collectinga fraction containing said homodimeric Fc fusion protein.

In another aspect, the present invention provides a homodimeric Fcfusion protein comprising a first monomer and a second monomer eachcomprising, from N- to C-terminal, a Fc domain-a first domain linker-anIL-12p35 subunit domain-a second domain linker-an IL-12p40 subunitdomain.

In some embodiments, the present invention provides a homodimeric Fcfusion protein, wherein said modifications promoting homodimerization ofsaid Fc domain are a set of amino acid substitutions selected from thegroup consisting of L368D/K370S; S364K; S364K/E357L; S364K/E357Q;T411E/K360E/Q362E; D401K; T366S/L368A/Y407V; T366W;T366S/L368A/Y407V/Y349C; and T366W/S354C, according to EU numbering.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said first domain linker and said second domainlinker have the same amino acid sequence.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said Fc domain has an additional set of aminoacid substitutions comprising Q295E/N384D/Q418E/N421D, according to EUnumbering.

In some embodiments, the present invention provides a homodimeric Fcfusion protein, wherein said Fc domain has an additional set of aminoacid substitutions selected from the group consisting of G236R/L328R,E233P/L234V/L235A/G236_/S239K, E233P/L234V/L235A/G236_/S239K/A327G,E233P/L234V/L235A/G236_/S267K/A327G, E233P/L234V/L235A/G236_, andE233P/L234V/L235A/G236_/S267K, according to EU numbering.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected form the group consisting of SEQ ID NO:3 (human IL-12 subunitbeta (IL-12p40) precursor sequence) and SEQ ID NO:4 (human IL-12 subunitbeta (IL-12p40) mature form sequence), and said IL-12p35 subunit has apolypeptide sequence selected from the group consisting of SEQ ID NO:1(human IL-12 subunit alpha (IL-12p35) precursor sequence) and SEQ IDNO:2 (human IL-12 subunit alpha (IL-12p35) mature form sequence).

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein each of said Fc domain further comprises aminoacid substitutions M428L/N434S.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit and/or said IL-12p35 subunit is a variant IL-12p35 subunit.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit is a variant IL-12p40subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex; and/or said IL-12p35 subunit is a variant IL-12p35subunit having altered affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or IL-12receptor complex.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidmodifications at amino acid residues selected from the group consistingof E3, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32,E33, D34, L40, D41, Q42, S43, E45, L47, T54, 155, Q56, K58, E59, F60,G61, D62, Q65, Y66, E73, K84, E86, D87, G88, 189, W90, D93, D97, K99,E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156,R159, D161, N162, K163, D166, D170, Q172, D174, A176, C177, P178, A179,A180, E181, S183, P185, E187, N200, S204, F206, R208, D209, D214, N218,Q220, N226, Q229, E231, E235, T242, P243, S245, Y246, F247, S248, C252S,Q256, K158, K260, E262, K264, D265, D270, N281, Q289, D290, R291, Y292,Y293, and E299.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has one or more amino acidsubstitutions selected from the group consisting of D18N, D18K, E32Q,E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N, E73Q,D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N,R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N,K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has amino acidsubstitutions selected from the group consisting ofN103D/N113D/N200D/N281D, Q42E/E45Q, E45Q/Q56E, Q42E/E59Q, Q56E/E59Q,Q42E/E45Q/Q56E, E45Q/Q56E/E59Q, E32Q/E59Q, D34N/E59K, D34N/E59K/K99E,D34K/E59K/K99E, E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q,E59Q/E187Q, S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E,E59Q/K260E, E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y,E59Y/K99Y, E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p40 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:57(IL-12p40(N103D)), ii) SEQ ID NO:58 (IL-12p40(N113D)), iii) SEQ ID NO:59(IL-12p40(N200D)), iv) SEQ ID NO:60 (IL-12p40(N281D)), v) SEQ ID NO:61(IL-12p40(N103D/N113D/N200D/N281D)), vi) SEQ ID NO:62 (IL-12p40(Q42E)),vii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)),ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi)SEQ ID NO:67 (IL-12p40(Q42E/E45Q)), xii) SEQ ID NO:68(IL-12p40(E45Q/Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E/E59Q)), xiv)SEQ ID NO:70 (IL-12p40(Q56E/E59Q)), xv) SEQ ID NO:71(IL-12p40(Q42E/E45Q/Q56E)), xvi) SEQ ID NO:72(IL-12p40(E45Q/Q56E/E59Q)), xvii) SEQ ID NO:73 (IL-12p40(D161N)), xviii)SEQ ID NO:74 (IL-12p40(E73Q)), xix) SEQ ID NO:75 (IL-12p40(Q144E)), xx)SEQ ID NO:76 (IL-12p40(E262Q)), xxi) SEQ ID NO:77 (IL-12p40(E100Q)),xxii) SEQ ID NO:78 (IL-12p40(D18N)), xxiii) SEQ ID NO:79(IL-12p40(E33Q)), xxiv) SEQ ID NO:80 (IL-12p40(Q229E)), xxv) SEQ IDNO:81 (IL-12p40(E235Q)), xxvi) SEQ ID NO:82 (IL-12p40(Q256N)), xxvii)SEQ ID NO:83 (IL-12p40(E299Q)), xxviii) SEQ ID NO:84 (IL-12p40(D87N)),xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii)IL-12p40(S43K), xxxiii) SEQ ID NO:379 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E)), xxxiv) SEQ ID NO:205(IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii)IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl)SEQ ID NO:206 (IL-12p40(E32Q/E59Q)), xli) SEQ ID NO:207(IL-12p40(D34N/E59Q)), xlii) SEQ ID NO:208 (IL-12p40(E59Q/E187Q)),xliii) SEQ ID NO:209 (IL-12p40(S43E/E59Q)), xliv) SEQ ID NO:210(IL-12p40(S43K/E49Q)), xlv) SEQ ID NO:211 (IL-12p40(E59Q/K163E)), xlvi)SEQ ID NO:212 (IL-12p40(E59Q/K99E)), xlvii) SEQ ID NO:213(IL-12p40(E59Q/K258E)), xlviii) SEQ ID NO:214 (IL-12p40(E59Q/K260E)),xlix) SEQ ID NO: 326 (IL-12p40 (D34N/E59K)), 1) SEQ ID NO: 325(IL-12p40(E59K/K99E)), li) SEQ ID NO: 339 (IL-12p40(D18K/E59K/K99E)),lii) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), liii) SEQ ID NO: 336(IL-12p40 (E59K/K99Y)), liv) SEQ ID NO: 335 (IL-12p40 (E59Y/K99E)), lv)SEQ ID NO: 338 (IL-12p40 (E45K/E59K/K99E)), lvi) SEQ ID NO: 340(IL-12p40 (E59K/K99E/Q144E)), lvii) SEQ ID NO: 341 (IL-12p40(E59K/K99E/Q144K)), lviii) SEQ ID NO: 342 (IL-12p40 (E59K/K99E/R159E)),lix) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), lx) SEQ ID NO: 344(IL-12p40 (D18K/E59K/K99E/K264E)), lxi) SEQ ID NO: 360 (IL-12p40(C252S)), lxii) SEQ ID NO: 361 (IL-12p40 (DI8K/E59K/K99E/C252S)), lxiii)SEQ ID NO: 362 (IL-12p40 (D18K/E59K/K99E/C252S/K264E)), lxiv) SEQ ID NO:363 (IL-12p40 (E59K/K99Y/C252S)), lxv) SEQ ID NO: 364 (IL-12p40(E59K/K99E/C252S/K264E)), lxvi) SEQ ID NO: 365 (IL-12p40(E59K/K99E/C252S)), lxvii) SEQ ID NO: 254 (IL-12p40 (N103D/N113D)),lxviii) SEQ ID NO: 255 (IL-12p40 (N103D/N200D)), lxix) SEQ ID NO: 256(IL-12p40 (N103D/N281D)), lxx) SEQ ID NO: 257 (IL-12p40 (N113D/N200D)),lxxi) SEQ ID NO: 258 (IL-12p40 (N113D/N281D)), lxxii) SEQ ID NO: 259(IL-12p40 (N200D/N281D)), lxxiii) SEQ ID NO: 260 (IL-12p40(N103D/N113D/N200D)), lxxiv) SEQ ID NO: 261 (IL-12p40(N103D/N113D/N281D)), lxxv) SEQ ID NO: 262 (IL-12p40(N103D/N200D/N281D)), lxxvi) SEQ ID NO: 263 (IL-12p40(N113D/N200D/N281D)), lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)),lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)), lxxix) SEQ ID NO: 266(IL-12p40 (N200Q)), lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)), lxxxi) SEQID NO: 268 (IL-12p40 (N103Q/N113Q)), lxxxii) SEQ ID NO: 269 (IL-12p40(N103Q/N200Q)), lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q/N281Q)),lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q/N200Q)), lxxxv) SEQ ID NO: 272(IL-12p40 (N113Q/N281Q)), lxxxvi) SEQ ID NO: 273 (IL-12p40(N200Q/N281Q)), lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q/N113Q/N200Q)),lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q/N113Q/N281Q)), lxxxix) SEQ IDNO: 276 (IL-12p40 (N103Q/N200Q/N281Q)), xc) SEQ ID NO: 277 (IL-12p40(N113Q/N200Q/N281Q)), xci) SEQ ID NO:278 (IL-12p40(N103Q/N113Q/N200Q/N281Q)), xcii) SEQ ID NO:327 (IL-12p40(D34N/E59K/K99E)), xciii) SEQ ID NO:328 (IL-12p40 (D34K/E59K/K99E)),xciv) SEQ ID NO:329 (IL-12p40 (E32Q/D34N/E59K/K99E)), xcv) SEQ ID NO:331(IL-12p40 (E32K/D34N/E59K/K99E)), xcvi) SEQ ID NO: 337 (IL-12p40(E59Y/K99Y)), xcvii) SEQ ID NO:366 (IL-12p40(E59K/K99E/N103Q/C252S/K264E)), xcviii) SEQ ID NO:367 (IL-12p40(E59K/K99E/N113Q/C252S/K264E)), xcix) SEQ ID NO:368 (IL-12p40(E59K/K99E/N200Q/C252S/K264E)), c) SEQ ID NO:369 (IL-12p40(E59K/K99E/N281Q/C252S/K264E)), ci) SEQ ID NO:370 (IL-12p40(E59K/K99E/N103Q/N113Q/C252S/K264E)), cii) SEQ ID NO:371 (IL-12p40(E59K/K99E/N103Q/N200Q/C252S/K264E)), ciii) SEQ ID NO:372 (IL-12p40(E59K/K99E/N103Q/N281Q/C252S/K264E)), civ) SEQ ID NO:373 (IL-12p40(E59K/K99E/N113Q/N200Q/C252S/K264E)), cv) SEQ ID NO:374 (IL-12p40(E59K/K99E/N113Q/N281Q/C252S/K264E)), cvi) SEQ ID NO:375 (IL-12p40(E59K/K99E/N200Q/N281Q/C252S/K264E)), cvii) SEQ ID NO:376 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E)), cviii) SEQ ID NO:377(IL-12p40 (E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E)), and cix) SEQ IDNO:378 (IL-12p40 (E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E)).

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidmodifications as amino acid residues selected from the group consistingof Q20, N21, Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63,L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97,L124, M125, Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163,D165, I171, R181, 1182, R183, V185, T186, D188, R189, V190, S192, Y193,N195, and A196.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has one or more amino acidsubstitutions selected from the group consisting of N21D, Q35D, E38Q,D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A,L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K,E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has amino acidsubstitutions selected from the group consisting of N71D/N85D/N195D,N151D/E153Q, N151D/D165N, Q130E/N151D, N151D/K158E, E79Q/N151D,D55Q/N151D, N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q, N71Q/N195Q,N85Q/N195Q, N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, and N85D/N195D.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said IL-12p35 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:113(IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115(IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D/N85D/N195D)), v) SEQID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii)SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(Q135E)),ix) SEQ ID NO:121 (IL-12p35(Q35D)), x) SEQ ID NO:122 (IL-12p35(Q146E)),xi) SEQ ID NO:123 (IL-12p35(N76D)), xii) SEQ ID NO:124(IL-12p35(E162Q)), xiii) SEQ ID NO:125 (IL-12p35(E163Q)), xiv)IL-12p35(N21D), xv) SEQ ID NO:333 (IL-12p35(D55Q)), xvi) IL-12p35(E79Q),xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx)SEQ ID NO:227 (IL-12p35(N151K)), xxi) SEQ ID NO:226 (IL-12p35(E153K)),xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO:225(IL-12p35(N151D/E153Q)), xxv) SEQ ID NO:228 (IL-12p35(N151D/D165N)),xxvi) SEQ ID NO:229 (IL-12p35(Q130E/N151D)), xxvii) SEQ ID NO:230(IL-12p35(N151D/K158E)), xxviii) SEQ ID NO:231 (IL-12p35(E79Q/N151D)),xxix) SEQ ID NO:232 (IL-12p35(D55Q/N151D)), xxx) SEQ ID NO:233(IL-12p35(N136D/N151D)), xxxi) SEQ ID NO:234 (IL-12p35(N21D/N151D)),xxxii) SEQ ID NO:235 (IL-12p35(E143Q/N151D)), xxxiii) SEQ ID NO: 345(IL-12p35(F96A)), xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)), xxxv) SEQ IDNO: 347 (IL-12p35(L89A)), xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)),xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)), xxxviii) SEQ ID NO: 350(IL-12p35(L75A)), xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)), xxxv) SEQ IDNO: 279 (IL-12p35 (N71Q)), xxxvi) SEQ ID NO: 280 (IL-12p35 (N85Q)),xxxvii) SEQ ID NO: 281 (IL-12p35 (N195Q)), xxxviii) SEQ ID NO: 282(IL-12p35 (N71Q/N85Q)), xxxix) SEQ ID NO: 283 (IL-12p35 (N71Q/N195Q)),xl) SEQ ID NO: 284 (IL-12p35 (N85Q/N195Q), xli) SEQ ID NO: 285 (IL-12p35(N71Q/N85Q/N195Q)), xlii) SEQ ID NO: 286 (IL-12p35 (N71D/N85D)), xliii)SEQ ID NO: 287 (IL-12p35 (N71D/N195D), xliv) SEQ ID NO: 288 (IL-12p35(N85D/N195D)), xlv) SEQ ID NO: 333 (IL-12p35 (D55Q)), and xlvi) SEQ IDNO: 334 (IL-12p35 (D55K)).

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said first and second monomers are XENP31289.

In some embodiments, the present invention provides a homodimeric Fcfusion protein wherein said first and second monomers are XENP31291.

In some embodiments, the present invention provides a homodimeric Fcfusion protein composition comprising a homodimeric Fc fusion proteinfor use in treating cancer in a subject.

In some embodiments, the present invention provides a one or morenucleic acids encoding a homodimeric Fc fusion protein.

In some embodiments, the present invention provides a host cellcomprising said one or more nucleic acids encoding a homodimeric Fcfusion protein.

In some embodiments, the present invention provides a method of making ahomodimeric Fc fusion protein, said method comprising culturing a hostcell under conditions whereby said homodimeric Fc fusion protein isproduced.

In some embodiments, the present invention provides a method ofpurifying a homodimeric Fc fusion protein, said method comprising: a)providing a composition comprising the homodimeric Fc fusion protein; b)loading said composition onto an ion exchange column; and c) collectinga fraction containing said homodimeric Fc fusion protein.

In another aspect, the present invention provides a method of treatingcancer in a patient in need thereof, the method comprising administeringa therapeutically effective amount of a heterodimeric or homodimeric Fcfusion protein according to any of the previous claims to said patient.

In some embodiments, the present invention provides a method of treatingcancer in a patient in need thereof, further comprising administering atherapeutically effective amount of a checkpoint blockade antibody.

In some further embodiments, the present invention provides a method oftreating cancer in a patient in need thereof, wherein said checkpointblockade antibody is selected from an anti-PD-1 antibody, an anti-PD-L1antibody, an anti-TIM3 antibody, an anti-TIGIT antibody, an anti-LAG3antibody, and an anti-CTLA-4 antibody.

In some further embodiments, the present invention provides a method oftreating cancer in a patient in need thereof, wherein said anti-PD-1antibody is nivolumab, pembrolizumab, or pidilizumab.

In some further embodiments, the present invention provides a method oftreating cancer in a patient in need thereof, wherein said anti-PD-L1antibody is atezolizumab, avelumab, or durbalumab.

In some further embodiments, the present invention provides a method oftreating cancer in a patient in need thereof, wherein the patientexhibits an increase in lymphocytes following administration.

In some further embodiments, the present invention provides a method oftreating cancer in a patient in need thereof, wherein the patientexhibits an increase in peripheral CD8+ T cells followingadministration.

In some embodiments, the present invention provides method of treatingcancer in a patient in need thereof wherein the IL-12 Fc fusion proteinis administered before the checkpoint inhibitor to increase checkpointexpression prior to treatment.

In some embodiments, the present invention provides a method of treatingcancer in a patient in need thereof wherein the IL-12 Fc fusion proteinis administered before the checkpoint inhibitor to increase checkpointexpression prior to treatment.

In another aspect, the present invention provides a method of inducing Tcell expansion in a patient in need thereof comprising administering atherapeutically effective amount of a heterodimeric or homodimeric Fcfusion protein according any of the previous claims to said patient.

In some further embodiments, the present invention provides a method ofinducing T cell expansion in a patient in need thereof, furthercomprising administering a therapeutically effective amount of acheckpoint blockade antibody.

In some further embodiments, the present invention provides a method ofinducing T cell expansion in a patient in need thereof, wherein saidcheckpoint blockade antibody is selected from an anti-PD-1 antibody, ananti-PD-L1 antibody, an anti-TIM3 antibody, an anti-TIGIT antibody, ananti-LAG3 antibody, and an anti-CTLA-4 antibody.

In some further embodiments, the present invention provides a method ofinducing T cell expansion in a patient in need thereof, wherein saidanti-PD-1 antibody is nivolumab, pembrolizumab, or pidilizumab.

In some other further embodiments, the present invention provides amethod of inducing T cell expansion in a patient in need thereof,wherein said anti-PD-L1 antibody is atezolizumab, avelumab, ordurbalumab.

In some further embodiments, the present invention provides a method ofinducing T cell expansion in a patient in need thereof, wherein the Tcell expansion is at least a 2-fold increase in T cells.

In some embodiments, the present invention provides a method of inducingT cell expansion in a patient in need thereof wherein the IL-12 Fcfusion protein is administered before the checkpoint inhibitor toincrease checkpoint expression prior to treatment.

In some embodiments, the present invention provides a method of inducingT cell expansion in a patient in need thereof wherein the IL-12 Fcfusion protein is administered before the checkpoint inhibitor toincrease checkpoint expression prior to treatment.

In one aspect the present invention provides an IL-12p40 subunit. Inanother aspect the present invention provides an IL-12p35 subunit. In afurther aspect the present invention provides a heterodimeric complexcomprising a) an IL-12p40 subunit and b) a IL-12p35 subunit.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depicts the sequences for IL-12 and its receptors.

FIG. 2A-2E depicts useful pairs of Fc heterodimerization variant sets(including skew and pI variants). Variants without a corresponding“monomer 2” are pI variants which can be used alone on either monomer.

FIG. 3 depicts a list of isosteric variant antibody constant regions andtheir respective substitutions. pI_(−) indicates lower pI variants,while pI_(+) indicates higher pI variants. These can be optionally andindependently combined with other heterodimerization variants of theinventions (and other variant types as well, as outlined herein.)

FIG. 4 depicts useful ablation variants that ablate FcγR binding(sometimes referred to as “knock outs” or “KO” variants). Generally,ablation variants are found on both monomers, although in some casesthey may be on only one monomer.

FIG. 5 shows particularly useful embodiments of “non-cytokine”components of the invention.

FIG. 6 depicts a number of exemplary domain linkers. In someembodiments, these linkers find use linking the IL-12p35 subunit, theIL-12p40 subunit, or the single-chain IL-12 complex to the N-terminus ofthe Fc region. In some embodiments, these linkers find use fusingIL-12p35 subunit to the IL-12p40 subunit in the single-chain IL-12complex. It is important to note that the scIL-12 complex can compriseeither IL-12p35 N-terminally linked to IL-12p40 or IL-12p40 N-terminallylinked to IL-12p35. Also, in some cases as described herein, the hingeportion of an Fc domain serves as a domain linker, which can be combinedwith any of these linkers as well.

FIG. 7A-7E shows the sequences of several useful IL-12-Fc fusionbackbones based on human IgG1, without the cytokine sequences (e.g. theIL-12p35 subunit, the IL-12p40 subunit, or the scIL-12 complex).Backbone 1 is based on human IgG1 (356E/358M allotype), and includesC220S on both chains, the S364K/E357Q:L368D/K370S skew variants, theQ295E/N384D/Q418E/N421D pI variants on the chain with L368D/K370S skewvariants and the E233P/L234V/L235A/G236del/S267K ablation variants onboth chains. Backbone 2 is based on human IgG1 (356E/358M allotype), andincludes C220S on both chains, the S364K:L368D/K370S skew variants, theQ295E/N384D/Q418E/N421D pI variants on the chain with L368D/K370S skewvariants and the E233P/L234V/L235A/G236del/S267K ablation variants onboth chains. Backbone 3 is based on human IgG1 (356E/358M allotype), andincludes C220S on both chains, the S364K:L368E/K370S skew variants, theQ295E/N384D/Q418E/N421D pI variants on the chain with L368E/K370S skewvariants and the E233P/L234V/L235A/G236del/S267K ablation variants onboth chains. Backbone 4 is based on human IgG1 (356E/358M allotype), andincludes C220S on both chains, the D401K:K360E/Q362E/T411E skewvariants, the Q295E/N384D/Q418E/N421D pI variants on the chain withK360E/Q362E/T411E skew variants and the E233P/L234V/L235A/G236del/S267Kablation variants on both chains. Backbone 5 is based on human IgG1(356D/358L allotype), and includes C220S on both chains, theS364K/E357Q:L368D/K370S skew variants, the Q295E/N384D/Q418E/N421D pIvariants on the chain with L368D/K370S skew variants and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains.Backbone 6 is based on human IgG1 (356E/358M allotype), and includesC220S on both chains, the S364K/E357Q:L368D/K370S skew variants,Q295E/N384D/Q418E/N421D pI variants on the chain with L368D/K370S skewvariants and the E233P/L234V/L235A/G236del/S267K ablation variants onboth chains, as well as an N297A variant on both chains. Backbone 7 isidentical to 6 except the mutation is N297S. Alternative formats forbackbones 6 and 7 can exclude the ablation variantsE233P/L234V/L235A/G236del/S267K in both chains. Backbone 8 is based onhuman IgG4, and includes the S364K/E357Q:L368D/K370S skew variants, theQ295E/N384D/Q418E/N421D pI variants on the chain with L368D/K370S skewvariants, as well as a S228P (EU numbering, this is S241P in Kabat)variant on both chains that ablates Fab arm exchange as is known in theart. Backbone 9 is based on human IgG2, and includes theS364K/E357Q:L368D/K370S skew variants, the Q295E/N384D/Q418E/N421D pIvariants on the chain with L368D/K370S skew variants. Backbone 10 isbased on human IgG2, and includes the S364K/E357Q:L368D/K370S skewvariants, the Q295E/N384D/Q418E/N421D pI variants on the chain withL368D/K370S skew variants as well as a S267K variant on both chains.Backbone 11 is identical to backbone 1, except it includes M428L/N434SXtend mutations. Backbone 12 is based on human IgG1 (356E/358Mallotype), and includes C220S on both identical chain, the theE233P/L234V/L235A/G236del/S267K ablation variants on both identicalchains. Backbone 13 is based on human IgG1 (356E/358M allotype), andincludes C220S on both chain, the S364K/E357Q:L368D/K370S skew variants,the P217R/P229R/N276K pI variants on the chain with S364K/E357Q skewvariants and the E233P/L234V/L235A/G236del/S267K ablation variants onboth chains.

As will be appreciated by those in the art and outlined below, thesesequences can be used with any IL-12-Fc fusion formats outlined herein,including but not limited to IL-12-heteroFc, heteroFc-IL-12, andscIL-12-Fc formats as schematically depicted in FIG. 8 . It should benoted that for heteroFc-IL-12 fusions, the backbones may furthercomprise deletion of K447. Additionally, any IL-12p35 and/or IL-12p40variants can be incorporated into these FIG. 7 backbones in anycombination.

Included within each of these backbones are sequences that are 90, 95,98 and 99% identical (as defined herein) to the recited sequences,and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional aminoacid substitutions (as compared to the “parent” of the Figure, which, aswill be appreciated by those in the art, already contain a number ofamino acid modifications as compared to the parental human IgG1 (or IgG2or IgG4, depending on the backbone). That is, the recited backbones maycontain additional amino acid modifications (generally amino acidsubstitutions) in addition to the skew, pI and ablation variantscontained within the backbones of this figure.

FIGS. 8A-8F depict illustrative formats for the IL-12-Fc fusion proteinsof the present invention. The N-terminal IL-12 heterodimeric Fc fusionor “IL-12-heteroFc” (FIG. 8A) format comprises the IL-12p40 subunitrecombinantly fused to the N-terminus of one side of a heterodimeric Fcand the IL-12p35 subunit recombinantly fused to N-terminus of the otherside of the heterodimeric Fc. The IL-12p35 and IL-12p40 subunits may belinked to their respective Fc chains by a domain linker. The C-terminalIL-12 heterodimeric Fc fusion or “heteroFc-IL-12” (FIG. 8B) formatcomprises the IL-12p40 subunit recombinantly fused to the C-terminus ofone side of a heterodimeric Fc and the IL-12p35 subunit recombinantlyfused to the C-terminus of the other side of the heterodimeric Fc. TheIL-12p35 and IL-12p40 subunits may be linked to their respective Fcchains by a domain linker. The N-terminal single-chain IL-12-Fc fusionor “scIL-12-Fc” (FIGS. 8C-D) format comprises a single-chain IL-12complex (or “scIL-12 complex”) recombinantly fused to the N-terminus ofone side of a heterodimeric Fc (optionally via a domain linker), withthe other side of the molecule being a “Fc-only” or “empty-Fc”heterodimeric Fc. The C-terminal single-chain IL-12-Fc fusion or“Fc-scIL-12” (FIGS. 8E-F) format comprises a scIL-12 complexrecombinantly fused to the C-terminus of one side of a heterodimeric Fc(optionally via a domain linker), with the other side of the moleculebeing a “Fc-only” or “empty-Fc” heterodimeric Fc. The scIL-12 complexcan comprise either IL-12p35 N-terminally linked to IL-12p40 or IL-12p40N-terminally linked to IL-12p35, optionally but generally with a domainlinker. The order of the two subunits in the scIL-12 complex may bedesignated as follows: “scIL-12(p40/p35)”, wherein the IL-12p40 subunitis N-terminally linked to the IL-12p35 subunit, or “scIL-12(p35/p40)”,wherein the IL-12p35 is N-terminally linked to the IL-12p40 subunit.

FIG. 9 depicts the sequences of XENP27201, an illustrative IL-12-Fcfusion protein of the “IL-12-heteroFc” format, that contains thewild-type IL-12p40 and wild-type IL-12p35 sequences. Linkers are doubleunderlined (although as will be appreciated by those in the art, thelinkers can be replaced by other linkers, some of which are depicted inFIG. 6 ), and slashes (/) indicate the border(s) between IL-12p35,IL-12p40, linkers, and Fc regions.

FIG. 10 depicts the sequences of XENP27202, an illustrative IL-12-Fcfusion protein of the “heteroFc-IL-12” format, that contains thewild-type IL-12p40 and wild-type IL-12p35 sequences. Linkers are doubleunderlined (although as will be appreciated by those in the art, thelinkers can be replaced by other linkers, some of which are depicted inFIG. 6 ), and slashes (/) indicate the border(s) between IL-12p35,IL-12p40, linkers, and Fc regions.

FIG. 11 depicts the sequences of XENP27203 and XENP27204, illustrativeIL-12-Fc fusion proteins of the “scIL-12-Fc” format, that contains thewild-type IL-12p40 and wild-type IL-12p35 sequences. Linkers are doubleunderlined (although as will be appreciated by those in the art, thelinkers can be replaced by other linkers, some of which are depicted inFIG. 6 ), and slashes (/) indicate the border(s) between IL-12p35,IL-12p40, linkers, and Fc regions.

FIG. 12A-12C depict A) chromatogram illustrating purification part 2 ofXENP27201 (anion exchange chromatography following protein Achromatography), and the purity and homogeneity of peak B isolated fromanion exchange separation as depicted in FIG. 12A in comparison to peakA as determined by B) analytical size-exclusion chromatography withmulti-angle light scattering (aSEC-MALS) and C) analytical anionexchange chromatography (analytical AIEX). FIG. 12B also depicts themolecular weight of protein species in peaks as determined bymulti-angle light scattering.

FIG. 13A-13C depicts A) chromatogram illustrating purification part 2 ofXENP27203 (anion exchange chromatography following protein Achromatography), and the purity and homogeneity of peak B isolated fromanion exchange separation as depicted in FIG. 13A in comparison to peakA as determined by B) analytical size-exclusion chromatography withmulti-angle light scattering (SEC-MALS) and C) analytical anion exchangechromatography. FIG. 13B also depicts the molecular weight of proteinspecies in peaks as determined by multi-angle light scattering.

FIG. 14A-14B depicts cartoon schematics for A) bivalent IL-12p40-Fcfusion and B) bivalent IL-12p35-Fc fusion. Each fusion comprises eitherIL-12p40 or IL-12p35 subunits recombinant fused to the N-terminus of ahomodimeric Fc. The subunits may have a domain linker between theirrespective C-terminus and the N-terminus of the Fc region.

FIG. 15 depicts the sequences of XENP27560, a bivalent IL-12p40-Fcfusion, and XENP27561, a bivalent IL-12p35-Fc fusion (cartoon schematicsdepicted in FIG. 14 ), that contains the wild-type IL-12p40 andwild-type IL-12p35 sequences. Linkers are double underlined (although aswill be appreciated by those in the art, the linkers can be replaced byother linkers, some of which are depicted in FIG. 6 ), and slashes (/)indicate the border(s) between IL-12p35, IL-12p40, linkers, and Fcregions.

FIG. 16A-16D depicts STAT4 phosphorylation on A) CD4⁺CD45RA⁻CD25^(hi) Tcells, B) CD8⁺CD45RA⁻CD25^(hi) T cells, C) γδ T cells, and D) NK cellsfollowing incubation of activated PBMCs with the indicated testarticles.

FIG. 17 depicts residues on IL-12p40 (based on IL-12p40 mature formsequence) predicted to contribute to the binding of IL-12p40 with IL-12receptors.

FIG. 18 depicts aspartic acid, glutamic acid, asparagine, and glutamineresidues on IL-12p40 (based on IL-12p40 mature form sequence) identifiedusing the QuaSAR package in MOE to have an ASA score (water accessiblesurface area calculated using a radius of 1.4 Å for the water moleculeand a polyhedral representation for each atom) of at least 19.

FIG. 19 depicts residues on IL-12p40 (based on IL-12p40 mature formsequence) predicted to be in contact with IL-23 receptors (based oncrystal structure deposited in the PDB with accession number 5MZV) aswell as the predicted contact type(s). “D” indicates contact predictedbased on proximity. “H” indicates contact predicted based on potentialhydrogen bond. “I” indicates contact predicted based on potential saltbridge. “A” indicates contact predicted based potential arene binding.

FIG. 20A-20I depicts sequences for illustrative IL-12p40 variantsdesigned with the view to reduce the affinity of the IL-12 heterodimericcomplex for the IL-12 receptors and/or remove putative glycosylationsites. Modified amino acids are underlined and in bold.

FIG. 21A-21J depicts the amino acid sequences for illustrative IL-12p40variants with Fc fusion partners. Domain linkers are double-underlined,and IL-12p40 variants are italicized.

FIG. 22 depicts aspartic acid, glutamic acid, asparagine, and glutamineresidues on IL-12p35 (based on IL-12p35 mature form sequence) identifiedusing the QuaSAR package in MOE to have an ASA score (water accessiblesurface area calculated using a radius of 1.4 Å for the water moleculeand a polyhedral representation for each atom) of at least 103.

FIG. 23A-23C depicts sequences for illustrative IL-12p35 variantsdesigned with the view to reduce the affinity of the IL-12 heterodimericcomplex for the IL-12 receptors and/or remove putative glycosylationsites. Modified amino acids are underlined and in bold.

FIG. 24A-24D depicts the amino acid sequences for illustrative IL-12p35variants with Fc fusion partners. Domain linkers are double-underlined,and IL-12p35 variants are italicized.

FIG. 25A-25Q depicts sequences for illustrative variant IL-12-Fc fusionsdesigned with the view to reduce the affinity of the IL-12-Fc fusionsfor IL-12 receptors or to remove putative glycosylation sites. Linkersare double underlined (although as will be appreciated by those in theart, the linkers can be replaced by other linkers, some of which aredepicted in FIG. 6 ), and slashes (/) indicate the border(s) betweenIL-12p35, IL-12p40, linkers, and Fc regions.

FIG. 26A-26B depicts STAT4 phosphorylation on A) CD4⁺CD45RA⁺CD25⁺ Tcells and B) CD8⁺CD45RA⁺CD25⁺ T cells following incubation of activatedPBMCs with IL-12-Fc fusions comprising IL-12p40 variants engineered withan aim to reduce affinity and potency

FIG. 27A-27B depicts STAT4 phosphorylation on A) CD4⁺CD45RA⁺CD25⁺ Tcells and B) CD8⁺CD45RA⁺CD25⁺ T cells following incubation of activatedPBMCs with IL-12-Fc fusions comprising IL-12p35 variants engineered withan aim to reduce affinity and potency

FIG. 28 depicts the EC50 (for STAT4 phosphorylation) as IL-12-Fc fusionscomprising IL-12p40 or IL-12p35 variants and the fold decrease in EC50relative to WT IL-12-Fc XENP27201. 27201-1 and 27201-2 represent twoseparately produced batches of XENP27201.

FIG. 29A-29B depicts sequences for illustrative IL-12p40 variantsdesigned with the view to reduce the affinity of the IL-12 heterodimericcomplex for the IL-12 receptors. Modified amino acids are underlined andin bold.

FIG. 30A-30D depicts the amino acid sequences for illustrative IL-12p40variants with Fc fusion partner. Domain linkers are double-underlined,and IL-12p40 variants are italicized.

FIG. 31A-31B depicts sequences for illustrative IL-12p35 variantsdesigned with the view to reduce the affinity of the IL-12 heterodimericcomplex for the IL-12 receptors. Modified amino acids are underlined andin bold.

FIG. 32A-32C depicts the amino acid sequences for illustrative IL-12p35variants with Fc fusion partners. Domain linkers are double-underlined,and IL-12p35 variants are italicized.

FIG. 33A-33P depicts sequences for illustrative variant IL-12-Fc fusionsdesigned with the view to reduce the affinity of the IL-12-Fc fusionsfor IL-12 receptors. Linkers are double underlined (although as will beappreciated by those in the art, the linkers can be replaced by otherlinkers, some of which are depicted in FIG. 6 ), and slashes (/)indicate the border(s) between IL-12p35, IL-12p40, linkers, and Fcregions.

FIGS. 34A and 34B depicts STAT4 phosphorylation on A) CD4⁺CD45RA⁺CD25⁺ Tcells and B) CD8⁺CD45RA⁺CD25⁺ T cells following incubation of activatedPBMCs with IL-12-Fc fusions comprising IL-12p40 variants engineered withan aim to reduce affinity and potency.

FIGS. 35A and 35B depicts STAT4 phosphorylation on A) CD4⁺CD45RA⁺CD25⁺ Tcells and B) CD8⁺CD45RA⁺CD25⁺ T cells following incubation of activatedPBMCs with IL-12-Fc fusions comprising IL-12p40 and/or IL-12p35 variantsengineered with an aim to reduce affinity and potency.

FIG. 36 depicts the EC50 (for STAT4 phosphorylation) of IL-12-Fc fusionscomprising IL-12p40 and/or IL-12p35 variants and the fold decrease inEC50 relative to WT IL-12-Fc XENP27201. The data show that potency wasreduced by up to 12-fold by an IL-12-Fc fusion comprising only a E59Ksubstitution in the IL-12p40 subunit.

FIG. 37A-37B depicts sequences for illustrative IL-12p40 variantsdesigned with the view to reduce the affinity of the IL-12 heterodimericcomplex for the IL-12 receptors. Modified amino acids are underlined andin bold.

FIG. 38 depicts sequences for illustrative IL-12p35 variants designedwith the view to reduce the affinity of the IL-12 heterodimeric complexfor the IL-12 receptors. Modified amino acids are underlined and inbold.

FIG. 39A-39I depicts sequences for illustrative variant IL-12-Fc fusionsdesigned with the view to reduce the affinity of the IL-12-Fc fusionsfor IL-12 receptors. Linkers are double underlined (although as will beappreciated by those in the art, the linkers can be replaced by otherlinkers, some of which are depicted in FIG. 6 ), and slashes (/)indicate the border(s) between IL-12p35, IL-12p40, linkers, and Fcregions.

FIG. 40A-40B depicts STAT4 phosphorylation on A) CD4⁺CD45RA⁺CD25⁺ Tcells and B) CD8⁺CD45RA⁺CD25⁺ T cells following incubation of activatedPBMCs with IL-12-Fc fusions comprising Il-12p40 and/or IL-12p35 variantsengineered with an aim to reduce affinity and potency.

FIG. 41 depicts the EC50 (for STAT4 phosphorylation) of IL-12-Fc fusionscomprising IL-12p40 and/or IL-12p35 variants and the fold decrease inEC50 relative to WT IL-12-Fc XENP27201. The data show that potency wasreduced by up to 100-fold.

FIG. 42A-42B depicts sequences for illustrative IL-12p40 variantsdesigned with the view to reduce the affinity of the IL-12 heterodimericcomplex for the IL-12 receptors. Modified amino acids are underlined andin bold.

FIG. 43 depicts sequences for illustrative IL-12p35 variants designedwith the view to reduce the affinity of the IL-12 heterodimeric complexfor the IL-12 receptors. Modified amino acids are underlined and inbold.

FIG. 44A-44K depicts sequences for illustrative variant IL-12-Fc fusionsdesigned with the view to reduce the affinity of the IL-12-Fc fusionsfor IL-12 receptors. Linkers are double underlined (although as will beappreciated by those in the art, the linkers can be replaced by otherlinkers, some of which are depicted in FIG. 6 ), and slashes (/)indicate the border(s) between IL-12p35, IL-12p40, linkers, and Fcregions.

FIG. 45A-45C depicts sequences for illustrative variant IL-12-Fc fusionsdesigned with the view to reduce the affinity of the IL-12-Fc fusionsfor IL-12 receptors, further engineered with Xtend Fc (M428L/N434S) forextending half-life. Linkers are double underlined (although as will beappreciated by those in the art, the linkers can be replaced by otherlinkers, some of which are depicted in FIG. 6 ), and slashes (/)indicate the border(s) between IL-12p35, IL-12p40, linkers, and Fcregions. It should be noted that these sequences are provided forillustrative purposes, and that any of the sequences depicted in theother Figures may also include Xtend Fc (M428L/N434S) for extendinghalf-life.

FIG. 46A-46D depict STAT4 phosphorylation on A) CD4⁺CD45RA⁺CD25⁺ Tcells, B) CD4⁺CD45RA⁻CD25⁺ T cells, C) CD8⁺CD45RA⁺CD25⁺ T cells, and D)CD8⁺CD45RA⁻CD25⁺ T cells following incubation of activated PBMCs withIL-12-Fc fusions comprising IL-12p40 and/or IL-12p35 variants engineeredwith an aim to reduce affinity and potency.

FIG. 47 depicts the EC50 (for STAT4 phosphorylation) of IL-12-Fc fusionscomprising IL-12p40 and/or IL-12p35 variants and the fold decrease inEC50 relative to WT IL-12-Fc XENP27201.

FIGS. 48A-48D depict illustrative formats for bivalent IL-12-Fc fusionproteins of the present invention. The bivalent N-terminal single-chain(FIGS. 48A-B) format comprises two identical monomers each comprising ascIL-12 complex recombinant fused to the N-terminus of a homodimeric Fcchain (optionally via a domain linker). The bivalent C-terminalsingle-chain (FIGS. 48C-D) format comprises two identical monomers eachcomprising a scIL-12 complex recombinant fused to the C-terminus of ahomodimeric Fc chain (optionally via a domain linker). The scIL-12complex can comprise either IL-12p35 N-terminally linked to IL-12p40 orIL-12p40 N-terminally linked to IL-12p35, optionally with a domainlinker. The order of the two subunits in the scIL-12 complex may bedesignated as follows: “scIL-12(p40/p35)”, wherein the IL-12p40 subunitis N-terminally linked to the IL-12p35 subunit, or “scIL-12(p35/p40)”,wherein the IL-12p35 is N-terminally linked to the IL-12p40 subunit.

FIG. 49 depicts the sequences for XENP31289 and XENP31291, illustrativeIL-12-Fc fusion proteins of the (scIL-12(p40/p35))₂-Fc format. XENP31289contains the wild-type IL-12p40 and wild-type IL-12p35 subunits.XENP31291 contains the IL-12p40(E59K/K99E) variant and wild-typeIL-12p35 subunits. Linkers are double underlined (although as will beappreciated by those in the art, the linkers can be replaced by otherlinkers, some of which are depicted in FIG. 6 ), and slashes (/)indicate the border(s) between IL-12p35, IL-12p40, linkers, and Fcregions.

FIG. 50 depicts the sequences for XENP31290, an illustrative IL-12-Fcfusion protein of the scIL-12(p40/p35)-Fc format, that contains theIL-12p40(E59K/K99E) variant and wild-type IL-12p35 subunits. Linkers aredouble underlined (although as will be appreciated by those in the art,the linkers can be replaced by other linkers, some of which are depictedin FIG. 6 ), and slashes (/) indicate the border(s) between IL-12p35,IL-12p40, linkers, and Fc regions.

FIGS. 51A-51D depict STAT4 phosphorylation on A) CD4⁺CD45RA⁺CD25⁺ Tcells, B) CD4⁺CD45RA⁻CD25⁺ T cells, C) CD8⁺CD45RA⁺CD25⁺ T cells, and D)CD8⁺CD45RA⁻CD25⁺ T cells following incubation of activated PBMCs withIL-12-Fc fusions in the scIL-12(p40/p35)-Fc and (scIL-12(p40/p35))₂-Fcformats with either WT IL-12p40 subunits or variant IL-12p40(E59K/K99E)subunits. The data show that the IL-12-Fc fusions in scIL-12(p40/p35)-Fcand (scIL-12(p40/p35))₂-Fc fusions comprising variantIL-12p40(E59K/K99E) subunits demonstrated reduced potency relative toIL-12-Fc fusions comprising WT IL-12p40 subunits.

FIG. 52 depicts the EC50 (for STAT4 phosphorylation) of IL-12-Fc fusionsin the scIL-12(p40/p35)-Fc and (scIL-12(p40/p35))₂-Fc formats witheither WT IL-12p40 subunits or variant IL-12p40(E59K/K99E) subunits.

FIG. 53 depicts the sequences for XENP16432, anti-PD-1 mAb based onnivolumab and IgG1 backbone with E233P/L234V/L235A/G236del/S267Kablation variant.

FIGS. 54A-54D depict the change in tumor volume (as determined bycaliper measurements) on A) Day 11, B) Day 13, and C) Day 15 as well asD) over time in pp65-MCF7 and huPBMC-engrafted NSG mice dosed with PBS,XENP16432 (a bivalent anti-PD-1 mAb), or XENP29952 (a reduced potencyIL-12-Fc fusion in the IL-12-heteroFc format comprising variantIL-12p40(E59K)). XENP29952 significantly enhanced anti-tumor activity byDay 11 as indicated by change in tumor volume (statistics performed onbaseline corrected data using unpaired t-test).

FIGS. 55A-55F depict A) CD45 cell, B) CD3⁺ T cell, C) CD4⁺ T cell, D)CD8⁺ T cell, E) NK cell counts as well as F) CD4⁺ T cell to CD8⁺ T cellratio in pp65-MCF7 and huPBMC-engrafted NSG mice on Day 14 followingPBMC-engraftment and first dose of PBS, XENP16432 (a bivalent anti-PD-1mAb), or XENP29952 (a reduced potency IL-12-Fc fusion in theIL-12-heteroFc format comprising variant IL-12p40(E59K)). XENP29952 hadsignificantly enhanced expansion of CD45+, CD3⁺ T cells, CD4⁺ T cells,CD8⁺ T cells, and NK cells by Day 14 in comparison to both PBS controland checkpoint blockade by XENP16432 (statistics performed onlog-transformed data using unpaired t-test).

FIGS. 56A-56D depict serum IFNγ concentrations on A) Day 7 and B) Day14, and serum CD25 concentrations on C) Day 7 and D) Day 14 in pp65-MCF7and huPBMC-engrafted NSG mice following PBMC-engraftment and first doseof PBS, XENP16432 (a bivalent anti-PD-1 mAb), or XENP29952 (a reducedpotency IL-12-Fc fusion in the IL-12-heteroFc format comprising variantIL-12p40(E59K)). XENP29952 significantly enhanced secretion of IFNγ andCD25 by Day 7 in comparison to checkpoint blockade by XENP16432(statistics performed on log-transformed date using unpaired t-test).

FIGS. 57A-57I depict change in body weight (as an indicator of GVHD) byA) Day 3, B) Day 6, C) Day 10, D) Day 13, E) Day 17, F) Day 20, G) Day24, and H) Day 27, as well as I) over time in huPBMC-engrafted NSG micedosed with PBS or XENP29952 (a reduced potency IL-12-Fc fusion in theIL-12-heteroFc format comprising variant IL-12p40(E59K)), XENP30597 (areduced potency IL-12-Fc fusion in the IL-12-heteroFc format comprisingvariant IL-12p40(E59K/K99E)), XENP31254 (a reduced potency IL-12-Fcfusion in the IL-12-heteroFc format comprising variantIL-12p40(D18K/E59K/K99E)), XENP31251 (a reduced potency IL-12-Fc fusionin the IL-12-heteroFc format comprising variant IL-12p40(E59K/K99Y)), orXENP31258 (a reduced potency IL-12-Fc fusion in the IL-12-heteroFcformat comprising variant IL-12p40(E59K/K99E/K264E)) at 0.3 or 0.03mg/kg. Notably, the IL-12-Fc fusion test articles induced varyingdegrees of GVHD which correlated with their in vitro potency.Additionally, the data show a dose response for the test articles (i.e.enhanced GVHD by 0.3 mg/kg vs. 0.03 mg/kg).

FIGS. 58A-58C depict PD-1 expression on CD8⁺ T cells (as an indicator ofactivation) in huPBMC-engrafted NSG mice on A) Day 7, B) Day 10, and C)Day 14 following PBMC-engraftment and first dose of PBS or XENP29952 (areduced potency IL-12-Fc fusion in the IL-12-heteroFc format comprisingvariant IL-12p40(E59K)), XENP30597 (a reduced potency IL-12-Fc fusion inthe IL-12-heteroFc format comprising variant IL-12p40(E59K/K99E)),XENP31254 (a reduced potency IL-12-Fc fusion in the IL-12-heteroFcformat comprising variant IL-12p40(D18K/E59K/K99E)), XENP31251 (areduced potency IL-12-Fc fusion in the IL-12-heteroFc format comprisingvariant IL-12p40(E59K/K99Y)), or XENP31258 (a reduced potency IL-12-Fcfusion in the IL-12-heteroFc format comprising variantIL-12p40(E59K/K99E/K264E)) at 0.3 or 0.03 mg/kg. Notably, the IL-12-Fcfusion test articles induced varying degrees of CD8⁺ T cell activationwhich correlated with their in vitro potency. Additionally, the datashow a dose response for the test articles (i.e. enhanced CD8⁺ T cellactivation by 0.3 mg/kg vs. 0.03 mg/kg).

FIGS. 59A-59C depict PD-1 expression on CD4⁺ T cells (as an indicator ofactivation) in huPBMC-engrafted NSG mice on A) Day 7, B) Day 10, and C)Day 14 following PBMC-engraftment and first dose of PBS or XENP29952 (areduced potency IL-12-Fc fusion in the IL-12-heteroFc format comprisingvariant IL-12p40(E59K)), XENP30597 (a reduced potency IL-12-Fc fusion inthe IL-12-heteroFc format comprising variant IL-12p40(E59K/K99E)),XENP31254 (a reduced potency IL-12-Fc fusion in the IL-12-heteroFcformat comprising variant IL-12p40(D18K/E59K/K99E)), XENP31251 (areduced potency IL-12-Fc fusion in the IL-12-heteroFc format comprisingvariant IL-12p40(E59K/K99Y)), or XENP31258 (a reduced potency IL-12-Fcfusion in the IL-12-heteroFc format comprising variantIL-12p40(E59K/K99E/K264E)) at 0.3 or 0.03 mg/kg. Notably, the IL-12-Fcfusion test articles induced varying degrees of CD4⁺ T cell activationwhich correlated with their in vitro potency. Additionally, the datashow a dose response for the test articles (i.e. enhanced CD4⁺ T cellactivation by 0.3 mg/kg vs. 0.03 mg/kg).

FIGS. 60A-60D depict serum concentration of IFNγ in huPBMC-engrafted NSGmice on Days A) 7, B) 10, C) 14 and D) 31 following PBMC-engraftment andfirst dose of PBS or XENP29952 (a reduced potency IL-12-Fc fusion in theIL-12-heteroFc format comprising variant IL-12p40(E59K)), XENP30597 (areduced potency IL-12-Fc fusion in the IL-12-heteroFc format comprisingvariant IL-12p40(E59K/K99E)), XENP31254 (a reduced potency IL-12-Fcfusion in the IL-12-heteroFc format comprising variantIL-12p40(D18K/E59K/K99E)), XENP31251 (a reduced potency IL-12-Fc fusionin the IL-12-heteroFc format comprising variant IL-12p40(E59K/K99Y)), orXENP31258 (a reduced potency IL-12-Fc fusion in the IL-12-heteroFcformat comprising variant IL-12p40(E59K/K99E/K264E)) at 0.3 or 0.03mg/kg. Notably, the IL-12-Fc fusion test articles induced varying levelsof IFNγ secretion which correlated with their in vitro potency.Additionally, the data show a dose response for the test articles (i.e.enhanced IFNγ secretion induced by 0.3 mg/kg vs. 0.03 mg/kg).

FIGS. 61A-61K depict STAT4 phosphorylation on A) CD4⁺CD45RA⁻CD25⁺ Tcells, B) CD4⁺CD45RA⁺CD25⁺ T cells, C) CD4⁺CD45RA⁺CD25⁻ T cells, D)CD4⁺CD45RA⁻CD25⁻ T cells, E) CD8⁺CD45RA⁻CD25⁺ T cells, F)CD8⁺CD45RA⁺CD25⁺ T cells, G) CD8⁺CD45RA⁺CD25⁻ T cells, H)CD8⁺CD45RA⁻CD25⁻ T cells, I) Tregs, J) γδ T cells, and K) CD56+NK cellsfollowing incubation of activated PBMCs (from a first donor; Donor 1)with recombinant human IL-12, XENP29952 (a reduced potency IL-12-Fcfusion in the IL-12-heteroFc format comprising variant IL-12p40(E59K)),XENP30597 (a reduced potency IL-12-Fc fusion in the IL-12-heteroFcformat comprising variant IL-12p40(E59K/K99E)), XENP31254 (a reducedpotency IL-12-Fc fusion in the IL-12-heteroFc format comprising variantIL-12p40(D18K/E59K/K99E)), XENP31251 (a reduced potency IL-12-Fc fusionin the IL-12-heteroFc format comprising variant IL-12p40(E59K/K99Y)), orXENP31258 (a reduced potency IL-12-Fc fusion in the IL-12-heteroFcformat comprising variant IL-12p40(E59K/K99E/K264E)). The data show apotency ladder with XENP29952 as the most potent variant, XENP31254 andXENP31258 as the least potent variants, and XENP30597 and XENP31251falling in between. Notably, the degree of GVHD and T cell activation asinduced by the reduced potency IL-12-Fc fusion variants in vivocorrelated with the in vitro potency.

FIGS. 62A-62D depict the change in tumor volume (as determined bycaliper measurements) on A) Day 14, B) Day 16, and C) Day 21 as well asD) over time in pp65-MCF7 and huPBMC-engrafted NSG mice dosed with PBS,XENP16432 (a bivalent anti-PD-1 mAb), XENP31258, XENP31251, or acombination of XENP31251 and XENP16432. XENP31258 significantly enhancedanti-tumor activity by Day 14, XENP31251 (alone or in combination withXENP16432) significantly enhanced anti-tumor activity by Day 16 incomparison to treatment with PBS; and XENP31251 in combination withXENP16432 significantly enhanced anti-tumor activity by Day 21 incomparison to treatment with XENP16432 alone (statistics performed onbaseline corrected data using Mann-Whitney test).

FIGS. 63A-63F depict A) CD45 cell, B) CD3⁺ T cell, C) CD4⁺ T cell, D)CD8⁺ T cell, E) NK cell counts as well as F) CD8⁺ T cell to CD4⁺ T cellratio in pp65-MCF7 and huPBMC-engrafted NSG mice on Day 14 followingPBMC-engraftment and first dose of PBS, XENP16432 (a bivalent anti-PD-1mAb), XENP31258, XENP31251, or a combination of XENP31251 and XENP16432.Notably, the data show that treatment with XENP31251 in combination withXENP16432 significantly enhanced lymphocyte expansion in comparison toeither XENP31251 or XENP16432 alone, indicating that IL-12-Fc fusionscombine productively with checkpoint blockade.

FIGS. 64A-64C depict sequences for illustrative variant IL-12-Fc fusionsdesigned with the view to reduce the affinity of the IL-12-Fc fusionsfor IL-12 receptors, further engineered with Xtend Fc (M428L/N434S) forextending half-life. Linkers are double underlined (although as will beappreciated by those in the art, the linkers can be replaced by otherlinkers, some of which are depicted in FIG. 6 ), and slashes (/)indicate the border(s) between Il-12p35, IL-12p40, linkers, and Fcregions. It should be noted that these sequences are provided forillustrative purposes, and that any of the sequences depicted in theother Figures may also include Xtend Fc (M428L/N434S) for extendinghalf-life.

FIGS. 65A-65C depict sequences for illustrative IL-12p40 variantsengineered with C252S with the view to remove the free cysteine (inaddition to expression and affinity/potency variants).

FIGS. 66A-66Q depict sequences for illustrative variant IL-12-Fc fusionsengineered with C252S in the IL-12p40 subunit with the view to removethe free cysteine(in addition to expression and affinity/potencyvariants). Linkers are double underlined (although as will beappreciated by those in the art, the linkers can be replaced by otherlinkers, some of which are depicted in FIG. 6 ), and slashes (/)indicate the border(s) between IL-12p35, IL-12p40, linkers, and Fcregions.

FIGS. 67A-67D depicts STAT4 phosphorylation on A) CD8⁺CD45RA⁻CD25⁺ Tcells, B) CD8⁺CD45RA⁺CD25⁺ T, C) CD4⁺CD45RA⁻CD25⁺ T, D) CD4⁺CD45RA⁺CD25⁺T, and E) NK cells following incubation of activated PBMCs with IL-12-Fccomprising IL-12p40 variants with or without additional engineering toremove free cysteine. The data show that most of the variants comprisingC252S in the IL-12p40 subunit demonstrated similar, albeit slightlyimproved, potency in comparison to the variants without the C252Smodification

DETAILED DESCRIPTION OF THE INVENTION I. Overview

The present invention is directed to novel heterodimeric fusion proteinconstructs containing IL-12 subunits and Fc domains. As noted above,IL-12 is composed of an α-chain (the p35 subunit; IL-12p35) and aβ-chain (the p40 subunit; IL-12p40) covalently linked to form thebiologically active IL-12 heterodimer. IL-12 exerts its cell signalingfunction through binding by binding to a dimeric IL-12 receptor complexcomposed of IL-12 receptor β1 (IL-12Rβ1) and IL-12 receptor β2(IL-12Rβ2) on T cells and inducing IFNγ secretion. However, the IL-12p40subunit can also exist as a homodimer which has been reported toantagonize IL-12 activity by competing for binding to IL-12 receptor.Accordingly, the present invention addresses the short half-life ofIL-12 and the potential formation of antagonistic IL12p40 homodimers byproviding IL-12-Fc fusion proteins, as well as novel IL-12 variants withdecreased potency. As generally shown in FIG. 8 , the heterodimericfusion proteins of the invention can take on a variety of conformations.

This application incorporates by reference 62/740,813 filed Oct. 3, 2018and 62/828,512 filed May 15, 2019, more specifically for FIG. 17 in62/740,813 and FIGS. 62A-62K and FIGS. 63A-63D in 62/828,512 as well asthe Figure Legends and the mention of these figures in the correspondingspecification.

II. Definitions

In order that the application may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “ablation” herein is meant a decrease or removal of binding and/oractivity. Thus for example, “ablating FcγR binding” means the Fc regionamino acid variant has less than 50% starting binding as compared to anFc region not containing the specific variant, with less than70-80-90-95-98% loss of binding being preferred, and in general, withthe binding being below the level of detectable binding in a Biacoreassay. Of particular use in the ablation of FcγR binding are those shownin FIG. 4 . However, unless otherwise noted, the Fc monomers of theinvention retain binding to the FcRn.

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. ADCC is correlated withbinding to FcγRIIIa; increased binding to FcγRIIIa leads to an increasein ADCC activity. As is discussed herein, many embodiments of theinvention ablate ADCC activity entirely.

By “modification” herein is meant an amino acid substitution, insertion,and/or deletion in a polypeptide sequence or an alteration to a moietychemically linked to a protein. For example, a modification may be analtered carbohydrate or PEG structure attached to a protein. By “aminoacid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. For clarity,unless otherwise noted, the amino acid modification is always to anamino acid coded for by DNA, e.g., the 20 amino acids that have codonsin DNA and RNA.

By “amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with a different amino acid. In particular, in someembodiments, the substitution is to an amino acid that is not naturallyoccurring at the particular position, either not naturally occurringwithin the organism or in any organism. For example, the substitutionE272Y or 272Y refers to a variant polypeptide, in this case an Fcvariant, in which the glutamic acid at position 272 is replaced withtyrosine. For clarity, a protein which has been engineered to change thenucleic acid coding sequence but not to change the starting amino acid(for example exchanging CGG (encoding arginine) to CGA (still encodingarginine) to increase host organism expression levels) is not an “aminoacid substitution”; that is, despite the creation of a new gene encodingthe same protein, if the protein has the same amino acid at theparticular position that it started with, it is not an amino acidsubstitution.

By “amino acid insertion” or “insertion” as used herein is meant theaddition of an amino acid residue or sequence at a particular positionin a parent polypeptide sequence. For example, −233E designates aninsertion of glutamic acid after position 233 and before position 234.Additionally, −233ADE or A233ADE designates an insertion of AlaAspGluafter position 233 and before position 234.

By “amino acid deletion” or “deletion” as used herein is meant theremoval of an amino acid residue or sequence at a particular position ina parent polypeptide sequence. For example, E233−, E233#, E233( ) orE233del designates a deletion of glutamic acid at position 233.Additionally, EDA233− or EDA233# designates a deletion of the sequenceGluAspAla that begins at position 233.

By “variant protein”, “protein variant”, or “variant” as used herein ismeant a protein that differs from that of a parent protein by virtue ofat least one modification. Protein variant may refer to the proteinitself, a composition comprising the protein, the amino acid sequencethat encodes it, or the DNA sequence that encodes it. Preferably, theprotein variant has at least one amino acid modification compared to theparent protein, e.g. from about one to about seventy amino acidmodifications, and preferably from about one to about five amino acidmodifications compared to the parent. The modification can be anaddition, deletion, or substitution. As described below, in someembodiments the parent polypeptide, for example an Fc parentpolypeptide, is a human wild type sequence, such as the Fc region fromIgG1, IgG2, IgG3 or IgG4. The protein variant sequence herein willpreferably possess at least about 80% identity with a parent proteinsequence, and most preferably at least about 90% identity, morepreferably at least about 95-98-99% identity.

As used herein, by “protein” is meant at least two covalently attachedamino acids, which includes proteins, polypeptides, oligopeptides andpeptides. When a biologically functional unit comprises two or moreproteins, each protein may be referred to as a “monomer” or as a“subunit” or as a “domain”; and the biologically functional molecule maybe referred to as a “complex”.

As used herein, by “protein” is meant at least two covalently attachedamino acids, which includes proteins, polypeptides, oligopeptides, andpeptides. When a biologically functional molecule or complex comprisestwo or more proteins, each protein may be referred to as a “monomer” oras a “subunit” or as a “domain”; and the biologically functionalmolecule may be referred to as a “complex” In some embodiments, the twoor more proteins of a functional complex are non-covalently attached. Insome embodiments, the term “monomer” refers to a polypeptide or proteincomprising one or more components, fragments, or subunits of aprotein(s), and the components, fragments, or subunits are covalentlyattached.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297 or N297) is a residue at position 297 in the humanantibody IgG1.

By “IgG subclass modification” or “isotype modification” as used hereinis meant an amino acid modification that converts one amino acid of oneIgG isotype to the corresponding amino acid in a different, aligned IgGisotype. For example, because IgG1 comprises a tyrosine and IgG2 aphenylalanine at EU position 296, a F296Y substitution in IgG2 isconsidered an IgG subclass modification.

The carboxy-terminal portion of each IgG chain defines a constant regionprimarily responsible for effector function. Kabat et al. collectednumerous primary sequences of the variable regions of heavy chains andlight chains. Based on the degree of conservation of the sequences, theyclassified individual primary sequences into the CDRs and the frameworkand made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5thedition, NIH publication, No. 91-3242, E. A. Kabat et al., entirelyincorporated by reference). Throughout the present specification, theKabat numbering system is generally used when referring to a residue inthe variable domain (approximately, residues 1-107 of the light chainvariable region and residues 1-113 of the heavy chain variable region)and the EU numbering system for Fc regions (e.g., Kabat et al., supra(1991)).

In the IgG subclass of immunoglobulins, there are several immunoglobulindomains in the heavy chain. By “immunoglobulin (Ig) domain” herein ismeant a region of an immunoglobulin having a distinct tertiarystructure. Of interest in the present invention are the heavy chaindomains, including, the constant heavy (CH) domains and the hingedomains. In the context of IgG antibodies, the IgG isotypes each havethree CH regions. Accordingly, “CH” domains in the context of IgG are asfollows: “CH1” refers to positions 118-215 according to the EU index asin Kabat. “Hinge” refers to positions 216-230 according to the EU indexas in Kabat. “CH2” refers to positions 231-340 according to the EU indexas in Kabat, and “CH3” refers to positions 341-447 according to the EUindex as in Kabat. As shown in Table 1, the exact numbering andplacement of the heavy chain domains can be different among differentnumbering systems. As shown herein and described below, the pI variantscan be in one or more of the CH regions, as well as the hinge region,discussed below.

Another type of Ig domain of the heavy chain is the hinge region. By“hinge” or “hinge region” or “antibody hinge region” or “immunoglobulinhinge region” herein is meant the flexible polypeptide comprising theamino acids between the first and second heavy chain constant domains ofan antibody. Structurally, the IgG CH1 domain ends at EU position 215,and the IgG CH2 domain begins at residue EU position 231. Thus for IgGthe antibody hinge is herein defined to include positions 216 (E216 inIgG1) to 230 (P230 in IgG1), wherein the numbering is according to theEU index as in Kabat. In some embodiments, for example in the context ofan Fc region, the hinge is included, generally referring to positions216-230. As noted herein, pI variants can be made in the hinge region aswell.

By “non-naturally occurring modification” as used herein is meant anamino acid modification that is not isotypic. For example, because noneof the IgGs comprise a serine at position 434, the substitution 434S inIgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered anon-naturally occurring modification.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids that are coded for by DNA andRNA.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toADCC, ADCP, and CDC.

By “IgG Fc ligand” or “Fc ligand” as used herein is meant a molecule,preferably a polypeptide, from any organism that binds to the Fc regionof an IgG antibody to form an Fc/Fc ligand complex. Fc ligands includebut are not limited to FcγRIs, FcγRIIs, FcγRIIIs, FcRn, C1q, C3, mannanbinding lectin, mannose receptor, staphylococcal protein A,streptococcal protein G, and viral FcγR. Fc ligands also include Fcreceptor homologs (FcRH), which are a family of Fc receptors that arehomologous to the FcγRs (Davis et al., 2002, Immunological Reviews190:123-136, entirely incorporated by reference). Fc ligands may includeundiscovered molecules that bind Fc. Particular IgG Fc ligands are FcRnand Fc gamma receptors.

By “Fc gamma receptor”, “FcγR” or “FcgammaR” as used herein is meant anymember of the family of proteins that bind the IgG antibody Fc regionand is encoded by an FcγR gene. In humans this family includes but isnot limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, andFcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypesH131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), andFcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirelyincorporated by reference), as well as any undiscovered human FcγRs orFcγR isoforms or allotypes. An FcγR may be from any organism, includingbut not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRsinclude but are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII(CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRsor FcγR isoforms or allotypes.

By “FcRn” or “neonatal Fc receptor” as used herein is meant a proteinthat binds the IgG antibody Fc region and is encoded at least in part byan FcRn gene. The FcRn may be from any organism, including but notlimited to humans, mice, rats, rabbits, and monkeys. As is known in theart, the functional FcRn protein comprises two polypeptides, oftenreferred to as the heavy chain and light chain. The light chain isbeta-2-microglobulin (42-microglobulin) and the heavy chain is encodedby the FcRn gene. Unless otherwise noted herein, FcRn or an FcRn proteinrefers to the complex of FcRn heavy chain with 42-microglobulin. Avariety of Fc variants can be used to increase binding to the FcRn, andin some cases, to increase serum half-life. In general, unless otherwisenoted, the Fc monomers of the invention retain binding to the FcRn (and,as noted below, can include amino acid variants to increase binding tothe FcRn).

By “parent polypeptide” as used herein is meant a starting polypeptidethat is subsequently modified to generate a variant. The parentpolypeptide may be a naturally occurring polypeptide, or a variant orengineered version of a naturally occurring polypeptide. Parentpolypeptide may refer to the polypeptide itself, compositions thatcomprise the parent polypeptide, or the amino acid sequence that encodesit.

By “Fc” or “Fc region” or “Fc domain” as used herein is meant thepolypeptide comprising the constant region of an antibody, in someinstances, excluding all of the first constant region immunoglobulindomain (e.g., CH1) or a portion thereof, and in some cases, optionallyincluding all or part of the hinge. For IgG, the Fc domain comprisesimmunoglobulin domains CH2 and CH3 (Cγ2 and Cγ3), and optionally all ora portion of the hinge region between CH1 (Cγ1) and CH2 (Cγ2). Thus, insome cases, the Fc domain includes, from N- to C-terminal, CH2-CH3 andhinge-CH2-CH3. In some embodiments, the Fc domain is that from IgG1,IgG2, IgG3 or IgG4, with IgG1 hinge-CH2-CH3 and IgG4 hinge-CH2-CH3finding particular use in many embodiments. Additionally, in the case ofhuman IgG1 Fc domains, frequently the hinge includes a C220S amino acidsubstitution. Furthermore, in the case of human IgG4 Fc domains,frequently the hinge includes a S228P amino acid substitution. Althoughthe boundaries of the Fc region may vary, the human IgG heavy chain Fcregion is usually defined to include residues E216, C226, or A231 to itscarboxyl-terminus, wherein the numbering is according to the EU index asin Kabat. In some embodiments, as is more fully described below, aminoacid modifications are made to the Fc region, for example to alterbinding to one or more FcγR or to the FcRn.

As will be appreciated by those in the art, the exact numbering andplacement of the heavy constant region domains can be different amongdifferent numbering systems. A useful comparison of heavy constantregion numbering according to EU and Kabat is as below, see Edelman etal., 1969, Proc Natl Acad Sci USA 63:78-85 and Kabat et al., 1991,Sequences of Proteins of Immunological Interest, 5th Ed., United StatesPublic Health Service, National Institutes of Health, Bethesda, entirelyincorporated by reference.

TABLE 1 EU Kabat Numbering Numbering CH1 118-215 114-223 Hinge 216-230226-243 CH2 231-340 244-360 CH3 341-447 361-478

“Fc variant” or “variant Fc” as used herein is meant a proteincomprising an amino acid modification in an Fc domain. The modificationcan be an addition, deletion, or substitution. The Fc variants of thepresent invention are defined according to the amino acid modificationsthat compose them. Thus, for example, N434S or 434S is an Fc variantwith the substitution for serine at position 434 relative to the parentFc polypeptide, wherein the numbering is according to the EU index.Likewise, M428L/N434S defines an Fc variant with the substitutions M428Land N434S relative to the parent Fc polypeptide. The identity of the WTamino acid may be unspecified, in which case the aforementioned variantis referred to as 428L/434S. It is noted that the order in whichsubstitutions are provided is arbitrary, that is to say that, forexample, 428L/434S is the same Fc variant as 434S/428L, and so on. Forall positions discussed in the present invention that relate toantibodies or derivatives and fragments thereof, unless otherwise noted,amino acid position numbering is according to the EU index. The EU indexor EU index as in Kabat or EU numbering scheme refers to the numberingof the EU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA63:78-85, hereby entirely incorporated by reference). The modificationcan be an addition, deletion, or substitution.

By “fusion protein” as used herein is meant covalent joining of at leasttwo proteins or protein domains. Fusion proteins may comprise artificialsequences, e.g. a domain linker, variant Fc domains, a variant IL-12p40subunit domain, a variant IL-12p35 subunit domain, etc. as describedherein. By “Fc fusion protein” or “immunoadhesin” herein is meant aprotein comprising an Fc region, generally linked (optionally through adomain linker, as described herein) to one or more different proteindomains. Accordingly, an “IL-12 Fc fusion” comprises an Fc region linked(optionally but usually through a domain linker) to an IL-12p40 subunit,an IL12p35 subunit and/or single-chain IL-12 complex (scIL-12), asdescribed herein. In some instances, two Fc fusion proteins can form ahomodimeric Fc fusion protein or a heterodimeric Fc fusion protein withthe latter being preferred in some instances. In some cases, one monomerof the heterodimeric Fc fusion protein comprises an Fc domain alone(e.g., an “empty Fc domain”) and the other monomer is an Fc fusion,comprising a variant Fc domain and one or two IL-12 subunit domains, asoutlined herein.

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index for antibody numbering.

By “strandedness” in the context of the monomers of the heterodimericproteins of the invention herein is meant that, similar to the twostrands of DNA that “match”, heterodimerization variants areincorporated into each monomer so as to preserve, create, and/or enhancethe ability to “match” to form heterodimers. For example, if some pIvariants are engineered into monomer A (e.g. making the pI higher), thensteric variants that are “charge pairs” that can be utilized as well donot interfere with the pI variants, e.g. the charge variants that make apI higher are put on the same “strand” or “monomer” to preserve bothfunctionalities. Similarly, for “skew” variants that come in pairs of aset as more fully outlined below, the skilled artisan will consider pIin deciding into which strand or monomer that incorporates one set ofthe pair will go, such that pI separation is maximized using the pI ofthe skews as well.

By “wild type or WT” herein is meant an amino acid sequence or anucleotide sequence that is found in nature, including allelicvariations. A WT protein has an amino acid sequence or a nucleotidesequence that has not been intentionally modified.

The heterodimeric proteins of the present invention are generallyisolated or recombinant. “Isolated,” when used to describe the variouspolypeptides disclosed herein, means a polypeptide that has beenidentified and separated and/or recovered from a cell or cell culturefrom which it was expressed. Ordinarily, an isolated polypeptide will beprepared by at least one purification step. An “isolated protein,”refers to a protein which is substantially free of other proteins from acell culture such as host cell proteins. “Recombinant” means theproteins are generated using recombinant nucleic acid techniques inexogeneous host cells.

“Percent (%) amino acid sequence identity” with respect to a proteinsequence is defined as the percentage of amino acid residues in acandidate sequence that are identical with the amino acid residues inthe specific (parental) sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.One particular program is the ALIGN-2 program outlined at paragraphs[0279] to [0280] of US Pub. No. 20160244525, hereby incorporated byreference.

The degree of identity between an amino acid sequence of the presentinvention (“invention sequence”) and the parental amino acid sequence iscalculated as the number of exact matches in an alignment of the twosequences, divided by the length of the “invention sequence,” or thelength of the parental sequence, whichever is the shortest. The resultis expressed in percent identity.

In some embodiments, two or more amino acid sequences are at least 50%,60%, 70%, 80%, or 90% identical. In some embodiments, two or more aminoacid sequences are at least 95%, 97%, 98%, 99%, or even 100% identical.

By “IL-12p40 subunit domain” herein is meant the β-chain (the p40subunit; IL-12p40). As discussed herein, the IL-12p40 subunit domain canbe a wild-type human sequence (e.g. SEQ ID NO: 3 from FIG. 1 ) or avariant thereof, as more fully discussed below (e.g. see FIGS. 20, 23and 29 , for example).

By “IL-12p35 subunit domain” herein is meant α-chain (the p35 subunit;IL-12p35). As discussed herein, the IL-12p35 subunit domain can be awild-type human sequence (e.g. SEQ ID NO: 1 from FIG. 1 ) or a variantthereof, as more fully discussed below (e.g. see FIGS. 20, 23 and 29 ,for example).

The IL-12 subunit domains of the invention, when associated together,specifically bind to a dimeric IL-12 receptor complex comprising IL-12receptor β1 and IL-12 receptor β2. The strength, or affinity, ofspecific binding can be expressed in terms of dissociation constant(K_(D)) of the interaction, wherein a smaller K_(D) represents greateraffinity and a larger K_(D) represents lower affinity. Bindingproperties can be determined by methods well known in the art such asbio-layer interferometry and surface plasmon resonance based methods,including Biacore and Octet methodologies. One such method entailsmeasuring the rates of antigen-binding site/antigen or receptor/ligandcomplex association and dissociation, wherein rates depend on theconcentration of the complex partners, the affinity of the interaction,and geometric parameters that equally influence the rate in bothdirections. Thus, both the association rate (k_(a)) and the dissociationrate (k_(d)) can be determined, and the ratio of k_(d)/k_(a) is equal tothe dissociation constant K_(D) (See Nature 361:186-187 (1993) andDavies et al. (1990) Annual Rev Biochem 59:439-473), both of which areincorporated by reference in their entirety for the methods therein.

Specific binding for a particular molecule can be exhibited, forexample, by a molecule having a K_(D) for a ligand (generally areceptor, in this case) of at least about 10⁻⁴ M, at least about 10⁻⁵ M,at least about 10⁻⁶ M, at least about 10⁻⁷ M, at least about 10⁻⁸ M, atleast about 10⁻⁹ M, alternatively at least about 10⁻¹⁰ M, at least about10⁻¹¹ M, at least about 10⁻¹² M, or greater. Typically, a molecule thatspecifically binds its receptor will have a K_(D) that is 20-, 50-,100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a controlmolecule relative to the receptor.

Also, specific binding for a particular molecule can be exhibited, forexample, by a molecule having a k_(a) or association rate for a ligandor receptor of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- ormore times greater for the receptor relative to a control.

By “fused” or “covalently linked” is herein meant that the components(e.g., an IL-12 subunit and an Fc domain) are linked by peptide bonds,either directly or indirectly via domain linkers, outlined herein.

As used herein, the term “single-chain” refers to a molecule comprisingamino acid domains linearly linked by peptide bonds. In certainembodiments, the biologically functional IL-12 is a single chain IL-12complex or “scIL-12”, i.e. the IL-12p35 subunit and the IL-12p40 subunitare fused to form a single peptide chain. In a particular suchembodiment, the C-terminus of the IL-12p35 subunit is connected to theN-terminus of the IL-12p40 subunit, designated “scIL-12(p35/p40)”. Inanother particular such embodiment, the C-terminus of the IL-12p40subunit is connected to the N-terminus of the IL-12p35 subunit,designated “scIL-12(p40/p35)”. Additionally, when these complexes arefurther fused to an Fc domain, they are still a “single chain”. Itshould be noted that these single chain constructs, where the p35 andp40 subunits are on the same amino acid chain, still are part of aheterodimeric complex containing two amino acid chains (e.g. as shown inFIGS. 8C and 8D, the scIL-12(p35/p40) component and the “empty arm” Fccomponent). That is, there are two single chains that make up theheterodimeric complex.

The fusion proteins of the invention can take on a variety of formats,including heterodimeric formats such as those depicted in FIG. 8 , aswell as homodimeric formats such as those depicted in FIG. 14 and FIG.48 .

III. Heterodimeric Fc Fusion Proteins

In some aspects, the present invention relates to heterodimeric Fcfusion proteins that include an Fc region, generally linked (optionallythrough a domain linker) to one or more different IL-12 protein domains.These heterodimeric fusion proteins can take on a large number ofdifferent formats, as are generally depicted in FIG. 8 . In one aspect,the heterodimeric Fc fusion protein is an IL-12 heterodimeric Fc fusionprotein that includes IL-12p40 and IL-12p35 subunits in differentorientations, such that they present together to bind to the IL-12receptor complex of IL-12Rβ1/β2. The Fc domains can be derived from IgGFc domains, e.g., IgG1, IgG2, IgG3 or IgG4 Fc domains, with IgG1 andIgG4 Fc domains finding particular use in the invention. As describedherein, IgG1 Fc domains may be used, often, but not always inconjunction with ablation variants to ablate effector function.Similarly, when low effector function is desired, IgG4 Fc domains may beused.

As described herein and known in the art, the heterodimeric proteins ofthe invention comprise different domains, which can be overlapping aswell. These domains include, but are not limited to, the Fc domain, theCH2 domain, the CH3 domain and the hinge domain, an IL-12p40 subunitdomain and an Il-12p35 subunit domain. As described herein, thesedomains are linked together in different formats, as generally outlinedin FIG. 8 .

In some of the embodiments herein, when a protein fragment, e.g.,IL-12p40 or IL-12p35 is attached to an Fc domain, it is the C-terminusof the protein fragment that is attached to all or part of the hinge ofthe Fc domain; for example, it is generally attached to the sequenceEPKS (SEQ ID NO: 466) which is the beginning of the IgG1 hinge. In otherof the embodiments herein, when a protein fragment, e.g., IL-12p40 orIL-12p35 is attached to an Fc domain, it is the N-terminus of theprotein fragment that is attached to the C-terminus of the CH3 domain.

In some of the constructs and sequences outlined herein of an Fc domainprotein, the C-terminus of the IL-12p40 or IL-12p35 protein fragment isattached to the N-terminus of a domain linker, the C-terminus of whichis attached to the N-terminus of a constant Fc domain (N-IL-12p40 orIL-12p35 protein fragment-linker-Fc domain-C) although that can beswitched (N-Fc domain-linker-IL-12p40 or IL-12p35 protein fragment-C).In other constructs and sequences outlined herein, the C-terminus of afirst protein fragment is attached to the N-terminus of a second proteinfragment, optionally via a domain linker, the C-terminus of the secondprotein fragment is attached to the N-terminus of a constant Fc domain,optionally via a domain linker. In yet another construct, the N-terminusof a first protein fragment is attached to the C-terminus of a secondprotein fragment, optionally via a domain linker, the N-terminus of thesecond protein fragment is attached to the C-terminus of a constant Fcdomain, optionally via a domain linker. In yet other constructs andsequences outlined herein, a constant Fc domain that is not attached toa first protein fragment or a second protein fragment is provided. Aheterodimer Fc fusion protein can contain two or more of the exemplarymonomeric Fc domain proteins described herein.

Accordingly, in some embodiments the present invention providesheterodimeric Fc fusion proteins that rely on the use of two differentheavy chain variant Fc sequences, that will self-assemble to form aheterodimeric Fc domain fusion polypeptide. In one embodiment,heterodimeric Fc fusion proteins contain at least two constant domainswhich can be engineered to produce heterodimers, such as pI engineering.Other Fc domains that can be used include fragments that contain one ormore of the CH1, CH2, CH3, and hinge domains of the invention that havebeen pI engineered. In particular, the formats depicted in FIGS. 8A-Fare heterodimeric Fc fusion proteins, meaning that the protein has twoassociated Fc sequences self-assembled into a heterodimeric Fc domainand at least one protein fragment (e.g., 1, 2 or more proteinfragments). In some cases, a first protein fragment is linked to a firstFc sequence and a second protein fragment is linked to a second Fcsequence. In some cases, the heterodimeric Fc fusion protein contains afirst protein fragment linked to a second protein fragment which islinked to a first Fc sequence, and a second Fc sequence that is notlinked to either the first or second protein fragments.

The present invention is directed to novel constructs to provideheterodimeric Fc fusion proteins that allow binding to one or morebinding partners, ligands or receptors. The heterodimeric Fc fusionconstructs are based on the self-assembling nature of the two Fc domainsof the heavy chains of antibodies, e.g., two “monomers” that assembleinto a “dimer”. Heterodimeric Fc fusions are made by altering the aminoacid sequence of each monomer as more fully discussed below. Thus, thepresent invention is generally directed to the creation of heterodimericFc fusion proteins which can co-engage binding partner(s) or ligand(s)or receptor(s) in several ways, relying on amino acid variants in theconstant regions that are different on each chain to promoteheterodimeric formation and/or allow for ease of purification ofheterodimers over the homodimers. There are a number of mechanisms thatcan be used to generate the heterodimers of the present invention. Inaddition, as will be appreciated by those in the art, these mechanismscan be combined to ensure high heterodimerization. Thus, amino acidvariants that lead to the production of heterodimers are referred to as“heterodimerization variants”. As discussed below, heterodimerizationvariants can include steric variants (e.g. the “knobs and holes” or“skew” variants described below and the “charge pairs” variantsdescribed below) as well as “pI variants”, which allows purification ofhomodimers away from heterodimers. As is generally described inWO2014/145806, hereby incorporated by reference in its entirety andspecifically as below for the discussion of “heterodimerizationvariants”, useful mechanisms for heterodimerization include “knobs andholes” (“KIH”; sometimes described herein as “skew” variants (seediscussion in WO2014/145806)), “electrostatic steering” or “chargepairs” as described in WO2014/145806, pI variants as described inWO2014/145806, and general additional Fc variants as outlined inWO2014/145806 and below.

In the present invention, there are several basic mechanisms that canlead to ease of purifying heterodimeric proteins and antibodies; onerelies on the use of pI variants, such that each monomer, andsubsequently each dimeric species, has a different pI, thus allowing theisoelectric purification of A-A, A-B and B-B dimeric proteins.Alternatively, some formats also allow separation on the basis of size.As is further outlined below, it is also possible to “skew” theformation of heterodimers over homodimers. Thus, a combination of stericheterodimerization variants and pI or charge pair variants findparticular use in the invention.

In general, embodiments of particular use in the present invention relyon sets of variants that include skew variants, that encourageheterodimerization formation over homodimerization formation, coupledwith pI variants, which increase the pI difference between the twomonomers and each dimeric species.

Additionally, as more fully outlined below, depending on the format ofthe heterodimer Fc fusion protein, pI variants can be either containedwithin the constant and/or Fc domains of a monomer, or domain linkerscan be used. That is, the invention provides pI variants that are on oneor both of the monomers, and/or charged domain linkers as well. Inaddition, additional amino acid engineering for alternativefunctionalities may also confer pI changes, such as Fc, FcRn and KOvariants.

In the present invention that utilizes pI as a separation mechanism toallow the purification of heterodimeric proteins, amino acid variantscan be introduced into one or both of the monomer polypeptides; that is,the pI of one of the monomers (referred to herein for simplicity as“monomer A”) can be engineered away from monomer B, or both monomer Aand B can be changed, with the pI of monomer A increasing and the pI ofmonomer B decreasing. As discussed, the pI changes of either or bothmonomers can be done by removing or adding a charged residue (e.g., aneutral amino acid is replaced by a positively or negatively chargedamino acid residue, e.g., glutamine to glutamic acid), changing acharged residue from positive or negative to the opposite charge (e.g.aspartic acid to lysine) or changing a charged residue to a neutralresidue (e.g., loss of a charge; lysine to serine.). A number of thesevariants are shown in the Figures.

Accordingly, this embodiment of the present invention provides forcreating a sufficient change in pI in at least one of the monomers suchthat heterodimers can be separated from homodimers. As will beappreciated by those in the art, and as discussed further below, thiscan be done by using a “wild type” heavy chain constant region and avariant region that has been engineered to either increase or decreaseits pI (wt A:B+ or wt A:B−), or by increasing one region and decreasingthe other region (A+:B− or A−:B+).

Thus, in general, a component of some embodiments of the presentinvention are amino acid variants in the constant regions that aredirected to altering the isoelectric point (pI) of at least one, if notboth, of the monomers of a dimeric protein by incorporating amino acidsubstitutions (“pI variants” or “pI substitutions”) into one or both ofthe monomers. The separation of the heterodimers from the two homodimerscan be accomplished if the pIs of the two monomers differ by as littleas 0.1 pH unit, with 0.2, 0.3, 0.4 and 0.5 or greater all finding use inthe present invention.

As will be appreciated by those in the art, the number of pI variants tobe included on each or both monomer(s) to get good separation willdepend in part on the starting pI of the components. That is, todetermine which monomer to engineer or in which “direction” (e.g., morepositive or more negative), the sequences of the Fc domains, and in somecases, the protein domain(s) linked to the Fc domain are calculated anda decision is made from there. As is known in the art, different Fcdomains and/or protein domains will have different starting pIs whichare exploited in the present invention. In general, as outlined herein,the pIs are engineered to result in a total pI difference of eachmonomer of at least about 0.1 logs, with 0.2 to 0.5 being preferred asoutlined herein.

Furthermore, as will be appreciated by those in the art and outlinedherein, in some embodiments, heterodimers can be separated fromhomodimers on the basis of size. As shown in the Figures, for example,several of the formats allow separation of heterodimers and homodimerson the basis of size.

In the case where pI variants are used to achieve heterodimerization, byusing the constant region(s) of Fc domains(s), a more modular approachto designing and purifying heterodimeric Fc fusion proteins is provided.Thus, in some embodiments, heterodimerization variants (including skewand purification heterodimerization variants) must be engineered. Inaddition, in some embodiments, the possibility of immunogenicityresulting from the pI variants is significantly reduced by importing pIvariants from different IgG isotypes such that pI is changed withoutintroducing significant immunogenicity. Thus, an additional problem tobe solved is the elucidation of low pI constant domains with high humansequence content, e.g. the minimization or avoidance of non-humanresidues at any particular position.

A side benefit that can occur with this pI engineering is also theextension of serum half-life and increased FcRn binding. That is, asdescribed in U.S. Ser. No. 13/194,904 (incorporated by reference in itsentirety), lowering the pI of antibody constant domains (including thosefound in antibodies and Fc fusions) can lead to longer serum retentionin vivo. These pI variants for increased serum half life also facilitatepI changes for purification.

In addition, it should be noted that the pI variants of theheterodimerization variants give an additional benefit for the analyticsand quality control process of Fc fusion proteins, as the ability toeither eliminate, minimize and distinguish when homodimers are presentis significant. Similarly, the ability to reliably test thereproducibility of the heterodimeric Fc fusion protein production isimportant.

A. Heterodimerization Variants

The present invention provides heterodimeric proteins, includingheterodimeric Fc fusion proteins in a variety of formats, which utilizeheterodimeric variants to allow for heterodimer formation and/orpurification away from homodimers. The heterodimeric fusion constructsare based on the self-assembling nature of the two Fc domains, e.g., two“monomers” that assemble into a “dimer”.

There are a number of suitable pairs of sets of heterodimerization skewvariants. These variants come in “pairs” of “sets”. That is, one set ofthe pair is incorporated into the first monomer and the other set of thepair is incorporated into the second monomer. It should be noted thatthese sets do not necessarily behave as “knobs in holes” variants, witha one-to-one correspondence between a residue on one monomer and aresidue on the other; that is, these pairs of sets form an interfacebetween the two monomers that encourages heterodimer formation anddiscourages homodimer formation, allowing the percentage of heterodimersthat spontaneously form under biological conditions to be over 90%,rather than the expected 50% (25% homodimer A/A:50% heterodimer A/B:25%homodimer B/B).

B. Steric Variants

In some embodiments, the formation of heterodimers can be facilitated bythe addition of steric variants. That is, by changing amino acids ineach heavy chain, different heavy chains are more likely to associate toform the heterodimeric structure than to form homodimers with the sameFc amino acid sequences. Suitable steric variants are included in theFIG. 29 of U.S. Ser. No. 15/141,350, all of which is hereby incorporatedby reference in its entirety, as well as in FIG. 2 .

One mechanism is generally referred to in the art as “knobs and holes”,referring to amino acid engineering that creates steric influences tofavor heterodimeric formation and disfavor homodimeric formation, asdescribed in U.S. Ser. No. 61/596,846, Ridgway et al., ProteinEngineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997 270:26;U.S. Pat. No. 8,216,805, all of which are hereby incorporated byreference in their entirety. The Figures identify a number of “monomerA-monomer B” pairs that rely on “knobs and holes”. In addition, asdescribed in Merchant et al., Nature Biotech. 16:677 (1998), these“knobs and hole” mutations can be combined with disulfide bonds to skewformation to heterodimerization.

An additional mechanism that finds use in the generation of heterodimersis sometimes referred to as “electrostatic steering” as described inGunasekaran et al., J. Biol. Chem. 285(25):19637 (2010), herebyincorporated by reference in its entirety. This is sometimes referred toherein as “charge pairs”. In this embodiment, electrostatics are used toskew the formation towards heterodimerization. As those in the art willappreciate, these may also have an effect on pI, and thus onpurification, and thus could in some cases also be considered pIvariants. However, as these were generated to force heterodimerizationand were not used as purification tools, they are classified as “stericvariants”. These include, but are not limited to, D221E/P228E/L368Epaired with D221R/P228R/K409R (e.g., these are “monomer” correspondingsets) and C220E/P228E/368E paired with C220R/E224R/P228R/K409R.

Additional monomer A and monomer B variants can be combined with othervariants, optionally and independently in any amount, such as pIvariants outlined herein or other steric variants that are shown in FIG.37 of US 2012/0149876, all of which are incorporated expressly byreference herein.

In some embodiments, the steric variants outlined herein can beoptionally and independently incorporated with any pI variant (or othervariants such as Fc variants, FcRn variants, etc.) into one or bothmonomers, and can be independently and optionally included or excludedfrom the proteins of the invention.

A list of suitable skew variants is found in FIG. 2 . Of particular usein many embodiments are the pairs of sets including, but not limited to,S364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K;T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L; K370S:S364K/E357Q; andT366S/L368A/Y407V:T366W (optionally including a bridging disulfide,T366S/L368A/Y407V/Y349C:T366W/S354C). In terms of nomenclature, the pair“S364K/E357Q:L368D/K370S” means that one of the monomers has the doublevariant set S364K/E357Q and the other has the double variant setL368D/K370S; as above, the “strandedness” of these pairs depends on thestarting pI.

C. pI (Isoelectric Point) Variants for Heterodimers

In general, as will be appreciated by those in the art, there are twogeneral categories of pI variants: those that increase the pI of theprotein (basic changes) and those that decrease the pI of the protein(acidic changes). As described herein, all combinations of thesevariants can be used: one monomer may be wild type, or a variant thatdoes not display a significantly different pI from wild-type, and theother can be either more basic or more acidic. Alternatively, eachmonomer may be changed, one to more basic and one to more acidic.

Preferred combinations of pI variants are shown in FIG. 30 of U.S. Ser.No. 15/141,350, all of which are herein incorporated by reference in itsentirety. As outlined herein and shown in the figures, these changes areshown relative to IgG1, but all isotypes can be altered this way, aswell as isotype hybrids. In the case where the heavy chain constantdomain is from IgG2-4, R133E and R133Q can also be used.

In one embodiment, a preferred combination of pI variants has onemonomer comprising 208D/295E/384D/418E/421D variants(N208D/Q295E/N384D/Q418E/N421D when relative to human IgG1) if one ofthe Fc monomers includes a CH1 domain. In some instances, the secondmonomer comprising a positively charged domain linker, including(GKPGS)₄ (SEQ ID NO: 462). In some cases, the first monomer includes aCH1 domain, including position 208. Accordingly, in constructs that donot include a CH1 domain (for example for heterodimeric Fc fusionproteins that do not utilize a CH1 domain on one of the domains), apreferred negative pI variant Fc set includes 295E/384D/418E/421Dvariants (Q295E/N384D/Q418E/N421D when relative to human IgG1).

In some embodiments, mutations are made in the hinge of the Fc domain,including positions 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, and 230. Thus, pI mutations and particularlysubstitutions can be made in one or more of positions 216-230, with 1,2, 3, 4 or 5 mutations finding use in the present invention. Again, allpossible combinations are contemplated, alone or with other pI variantsin other domains.

Specific substitutions that find use in lowering the pI of hinge domainsinclude, but are not limited to, a deletion at position 221, anon-native valine or threonine at position 222, a deletion at position223, a non-native glutamic acid at position 224, a deletion at position225, a deletion at position 235 and a deletion or a non-native alanineat position 236. In some cases, only pI substitutions are done in thehinge domain, and in others, these substitution(s) are added to other pIvariants in other domains in any combination.

In some embodiments, mutations can be made in the CH2 region, includingpositions 233, 234, 235, 236, 274, 296, 300, 309, 320, 322, 326, 327,334 and 339. It should be noted that changes in 233-236 can be made toincrease effector function (along with 327A) in the IgG2 backbone.Again, all possible combinations of these 14 positions can be made;e.g., a pI antibody may have 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 CH2 pIsubstitutions.

Specific substitutions that find use in lowering the pI of CH2 domainsinclude, but are not limited to, a non-native glutamine or glutamic acidat position 274, a non-native phenylalanine at position 296, anon-native phenylalanine at position 300, a non-native valine atposition 309, a non-native glutamic acid at position 320, a non-nativeglutamic acid at position 322, a non-native glutamic acid at position326, a non-native glycine at position 327, a non-native glutamic acid atposition 334, a non-native threonine at position 339, and all possiblecombinations within CH2 and with other domains.

In this embodiment, the mutations can be independently and optionallyselected from position 355, 359, 362, 384, 389,392, 397, 418, 419, 444and 447. Specific substitutions that find use in lowering the pI of CH3domains include, but are not limited to, a non-native glutamine orglutamic acid at position 355, a non-native serine at position 384, anon-native asparagine or glutamic acid at position 392, a non-nativemethionine at position 397, a non-native glutamic acid at position 419,a non-native glutamic acid at position 359, a non-native glutamic acidat position 362, a non-native glutamic acid at position 389, anon-native glutamic acid at position 418, a non-native glutamic acid atposition 444, and a deletion or non-native aspartic acid at position447.

D. Isotypic Variants

In addition, many embodiments of the invention rely on the “importation”of pI amino acids at particular positions from one IgG isotype intoanother, thus reducing or eliminating the possibility of unwantedimmunogenicity being introduced into the variants. A number of these areshown in FIG. 21 of US Publ. App. No. 2014/0370013, hereby incorporatedby reference. That is, IgG1 is a common isotype for therapeuticantibodies for a variety of reasons, including high effector function.However, the heavy constant region of IgG1 has a higher pI than that ofIgG2 (8.10 versus 7.31). By introducing IgG2 residues at particularpositions into the IgG1 backbone, the pI of the resulting monomer islowered (or increased) and additionally exhibits longer serum half-life.For example, IgG1 has a glycine (pI 5.97) at position 137, and IgG2 hasa glutamic acid (pI 3.22); importing the glutamic acid will affect thepI of the resulting protein. As is described below, a number of aminoacid substitutions are generally required to significantly affect the pIof the variant Fc fusion protein. However, it should be noted asdiscussed below that even changes in IgG2 molecules allow for increasedserum half-life.

In other embodiments, non-isotypic amino acid changes are made, eitherto reduce the overall charge state of the resulting protein (e.g., bychanging a higher pI amino acid to a lower pI amino acid), or to allowaccommodations in structure for stability, etc. as is more furtherdescribed below.

In addition, by pI engineering both the heavy and light constantdomains, significant changes in each monomer of the heterodimer can beseen. As discussed herein, having the pIs of the two monomers differ byat least 0.5 can allow separation by ion exchange chromatography orisoelectric focusing, or other methods sensitive to isoelectric point.

E. Calculating pI

The pI of each monomer can depend on the pI of the variant heavy chainconstant domain and the pI of the total monomer, including the variantheavy chain constant domain and the fusion partner. Thus, in someembodiments, the change in pI is calculated on the basis of the variantheavy chain constant domain, using the chart in the FIG. 19 of US Publ.App. No. 2014/0370013. As discussed herein, which monomer to engineer isgenerally decided by the inherent pI of each monomer.

F. pI Variants that Also Confer Better FcRn In Vivo Binding

In the case where the pI variant decreases the pI of the monomer, theycan have the added benefit of improving serum retention in vivo.

Although still under examination, Fc regions are believed to have longerhalf-lives in vivo, because binding to FcRn at pH 6 in an endosomesequesters the Fc (Ghetie and Ward, 1997 Immunol Today. 18(12): 592-598,entirely incorporated by reference). The endosomal compartment thenrecycles the Fc to the cell surface. Once the compartment opens to theextracellular space, the higher pH, ˜7.4, induces the release of Fc backinto the blood. In mice, Dall' Acqua et al. showed that Fc mutants withincreased FcRn binding at pH 6 and pH 7.4 actually had reduced serumconcentrations and the same half-life as wild-type Fc (Dall' Acqua etal. 2002, J. Immunol. 169:5171-5180, entirely incorporated byreference). The increased affinity of Fc for FcRn at pH 7.4 is thoughtto forbid the release of the Fc back into the blood. Therefore, the Fcmutations that will increase Fc's half-life in vivo will ideallyincrease FcRn binding at the lower pH while still allowing release of Fcat higher pH. The amino acid histidine changes its charge state in thepH range of 6.0 to 7.4. Therefore, it is not surprising to find Hisresidues at important positions in the Fc/FcRn complex.

There are a number of Fc substitutions that find use in increasedbinding to the FcRn and increased serum half-life, as specificallydisclosed in U.S. Ser. No. 12/341,769, hereby incorporated by referencein its entirety, including, but not limited to, 434A, 428L, 308F, 259I,428L/434S, 259I/308F, 436I/428L, 436I, 434S, 252Y/428L, 252Y/434S,428L/434S, 436V/428L and 259I/308F/428L.

G. Additional Fc Variants for Additional Functionality

In addition to pI amino acid variants, there are a number of useful Fcamino acid modification that can be made for a variety of reasons,including, but not limited to, altering binding to one or more FcγR,altered binding to FcRn, etc.

Accordingly, the proteins of the invention can include amino acidmodifications, including the heterodimerization variants outlinedherein, which includes the pI variants and steric variants. Each set ofvariants can be independently and optionally included or excluded fromany particular heterodimeric protein.

H. FcγR Variants

Accordingly, there are a number of useful Fc substitutions that can bemade to alter binding to one or more of the Fcγ receptors. Substitutionsthat result in increased binding as well as decreased binding can beuseful. For example, it is known that increased binding to FcγRIIIaresults in increased ADCC (antibody dependent cell-mediatedcytotoxicity; the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell). Similarly, decreasedbinding to FcγRIIb (an inhibitory receptor) can be beneficial as well insome circumstances. Amino acid substitutions that find use in thepresent invention include those listed in U.S. Ser. No. 11/124,620(particularly FIG. 41), Ser. Nos. 11/174,287, 11/396,495, 11/538,406,all of which are expressly incorporated herein by reference in theirentirety and specifically for the variants disclosed therein. Particularvariants that find use include, but are not limited to, 236A, 239D,239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E,239D/332E/330Y, 239D, 332E/330L, 243A, 243L, 264A, 264V and 299T.

In addition, amino acid substitutions that increase affinity for FcγRIIccan also be included in the Fc domain variants outlined herein. Thesubstitutions described in, for example, U.S. Ser. Nos. 11/124,620 and14/578,305 are useful.

I. Ablation Variants

Similarly, another category of functional variants are “FcγR ablationvariants” or “Fc knock out (FcKO or KO)” variants. In these embodiments,for some therapeutic applications, it is desirable to reduce or removethe normal binding of the Fc domain to one or more or all of the Fcγreceptors (e.g., FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa, etc.) to avoidadditional mechanisms of action. That is, for example, in manyembodiments, particularly in the use of immunomodulatory proteins, it isdesirable to ablate FcγRIIIa binding to eliminate or significantlyreduce ADCC activity such that one of the Fc domains comprises one ormore Fcγ receptor ablation variants. These ablation variants aredepicted in FIG. 31 of U.S. Ser. No. 15/141,350, all of which are hereinincorporated by reference in its entirety, and each can be independentlyand optionally included or excluded, with preferred aspects utilizingablation variants selected from the group consisting of G236R/L328R,E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S267K,E233P/L234V/L235A/G236del/S239K/A327G,E233P/L234V/L235A/G236del/S267K/A327G and E233P/L234V/L235A/G236del,according to the EU index. It should be noted that the ablation variantsreferenced herein ablate FcγR binding but generally not FcRn binding.

J. Combination of Heterodimeric and Fc Variants

As will be appreciated by those in the art, all of the recitedheterodimerization variants (including skew and/or pI variants) can beoptionally and independently combined in any way, as long as they retaintheir “strandedness” or “monomer partition”. In addition, all of thesevariants can be combined into any of the heterodimerization formats.

In the case of pI variants, while embodiments finding particular use areshown in the Figures, other combinations can be generated, following thebasic rule of altering the pI difference between two monomers tofacilitate purification.

In addition, any of the heterodimerization variants, skew and pI, mayalso be independently and optionally combined with Fc ablation variants,Fc variants, FcRn variants, as generally outlined herein.

In addition, a monomeric Fc domain can comprise a set of amino acidsubstitutions that includes C220S/S267K/L368D/K370S orC220S/S267K/S364K/E357Q.

In addition, the heterodimeric Fc fusion proteins can comprise skewvariants (e.g., a set of amino acid substitutions as shown in FIGS.1A-1C of U.S. Ser. No. 15/141,350, all of which are herein incorporatedby reference in its entirety), with particularly useful skew variantsbeing selected from the group consisting of S364K/E357Q:L368D/K370S;L368D/K370S:S364K; L368E/K370S:S364K; T411T/E360E/Q362E:D401K;L368D/K370S:S364K/E357L; K370S:S364K/E357Q; T366S/L368A/Y407V:T366W; andT366S/L368A/Y407V:T366W (optionally including a bridging disulfide,T366S/L368A/Y407V/Y349C:T366W/S354C), optionally ablation variants,optionally charged domain linkers; and optionally pI variants.

In some embodiments, the Fc domain comprises one or more amino acidsubstitutions selected from the group consisting of: 236R, S239D, S239E,F243L, M252Y, V259I, S267D, S267E, S67K, S298A, V308F, L328F, L328R,330L, I332D, I332E, M428L, N434A, N434S, 236R/L328R, S239D/I332E,236R/L328F, V259I/V308F, S267E/L328F, M428L/N43S, Y436I/M428L,N436V/M428L, V436I/N434S, Y436V/N434S, S239D/I332E/330L,M252Y/S54T/T256E, V259I/V308F/M428L, E233P/L234V/L235A/G236_/S239K,E233P/L234V/L235A/G236_/S239K/A327G,E233P/L234V/L235A/G236_/S267K/A327G, E233P/L234V/L235A/G236_, andE233P/L234V/L235A/G236_/S267K according to EU index.

In one embodiment, a particular combination of skew and pI variants thatfinds use in the present invention is T366S/L368A/Y407V:T366W(optionally including abridging disulfide,T366S/L368A/Y407V/Y349C:T366W/S354C) with one monomer comprisingQ295E/N384D/Q418E/N481D and the other a positively charged domainlinker. As will be appreciated in the art, the “knobs in holes” variantsdo not change pI, and thus can be used on either monomer.

IV. Homodimeric Fc Fusion Proteins

In some aspects, the present invention relates to homodimeric Fc fusionproteins that include an Fc region, generally linked (optionally througha domain linker) to one or more different protein domains. These formatsare generally shown in FIG. 14 and FIG. 48 . In one aspect, thehomodimeric Fc fusion protein is an IL-12 homodimeric Fc fusion proteinthat includes IL-12p40 and IL-12p35 subunits in different orientations,such that they present together to bind to the IL-12 receptor complex ofIL-12Rβ1/β2. The Fc domains can be derived from IgG Fc domains, e.g.,IgG1, IgG2, IgG3 or IgG4 Fc domains, with IgG1 and IgG4 Fc domainsfinding particular use in the invention. As described herein, IgG1 Fcdomains may be used, often, but not always in conjunction with ablationvariants to ablate effector function. Similarly, when low effectorfunction is desired, IgG4 Fc domains may be used.

In one aspect, a homodimeric Fc fusion protein comprises first monomerand a second monomer comprising, from N- to C-terminal, IL-12p40subunit-optional linker-IL-12p35 subunit-optional linker-Fc domain. Insome embodiments, a homodimeric Fc fusion protein comprises a firstmonomer and a second monomer comprising, from N- to C-terminal, IL-12p40subunit-linker-IL-12p35 subunit-linker-Fc domain. An example of anembodiment is provided in FIG. 48A.

In another aspect, a homodimeric Fc fusion protein comprises a firstmonomer and a second monomer comprising, from N- to C-terminal, IL-12p35subunit-optional linker-IL-12p40 subunit-optional linker-Fc domain. Insome embodiments, a homodimeric Fc fusion protein comprises a firstmonomer and a second monomer comprising, from N- to C-terminal, IL-12p35subunit-linker-IL-12p40 subunit-linker-Fc domain. An example of anembodiment is provided in FIG. 48B.

In another aspect, a homodimeric Fc fusion protein comprises a firstmonomer and a second monomer comprising, from N- to C-terminal, Fcdomain-optional linker-IL-12p40 subunit-optional linker-IL-12p35subunit. In some embodiments, a homodimeric Fc fusion protein comprisinga first monomer and a second monomer comprises, from N- to C-terminal,Fc domain-linker-IL-12p40 subunit-linker-IL-12p35 subunit. An example ofan embodiment is provided in FIG. 48C.

In another aspect, a homodimeric Fc fusion protein comprises a firstmonomer and a second monomer comprising, from N- to C-terminal, Fcdomain-optional linker-IL-12p35 subunit-optional linker-IL-12p40subunit. In some embodiments, a homodimeric Fc fusion protein comprisinga first monomer and a second monomer comprises, from N- to C-terminal,Fc domain-linker-IL-12p35 subunit-linker-IL-12p40 subunit. An example ofan embodiment is provided in FIG. 48D.

In some embodiments, the bivalent IL-12p40-Fc format provides ahomodimeric Fc fusion protein comprising: a) a first monomer comprising,from N- to C-terminal: i) a IL-12p40 domain protein; ii) an optionalfirst domain linker; iii) a first variant Fc domain protein; and b) asecond monomer comprising, from N- to C-terminal: i) a IL-12p40 domainprotein; ii) optionally a second domain linker; iii) a second variant Fcdomain protein.

In some embodiments, the bivalent IL-12p35-Fc format provides ahomodimeric Fc fusion protein comprising: a) a first monomer comprising,from N- to C-terminal: i) a IL-12p35 domain protein; ii) an optionalfirst domain linker; iii) a first variant Fc domain protein; and b) asecond monomer comprising, from N- to C-terminal: i) a IL-12p35 domainprotein; ii) optionally a second domain linker; iii) a second variant Fcdomain protein.

In some embodiments, the (scIL-12(p40/p35))₂-Fc format provides ahomodimeric Fc fusion protein comprising: a) a first monomer comprising,from N- to C-terminal: i) a IL-12p40 domain protein; ii) an optionalfirst domain linker; iii) a IL-12p35 domain protein; iv) a optionalsecond domain linker; v) a first variant Fc domain protein; and b) asecond monomer comprising, from N- to C-terminal: i) a IL-12p40 domainprotein; ii) optionally a third domain linker; iii) a IL-12p35 domainprotein; iv) optionally a fourth domain linker; v) a second variant Fcdomain protein.

In some embodiments, the (scIL-12(p35/p40))₂-Fc format provides ahomodimeric Fc fusion protein comprising: a) a first monomer comprising,from N- to C-terminal: i) a IL-12p35 domain protein; ii) an optionalfirst domain linker; iii) a IL-12p40 domain protein; iv) a optionalsecond domain linker; v) a first variant Fc domain protein; and b) asecond monomer comprising, from N- to C-terminal: i) a IL-12p35 domainprotein; ii) optionally a third domain linker; iii) a IL-12p40 domainprotein; iv) optionally a fourth domain linker; v) a second variant Fcdomain protein.

In some embodiments, the Fc-(scIL-12(p40/p35))₂ format provides ahomodimeric Fc fusion protein comprising: a) a first monomer comprising,from N- to C-terminal: i) a first variant Fc domain protein; ii) anoptional first domain linker; iii) a IL-12p40 domain protein; iv) anoptional second domain linker; v) a IL-12p35 domain protein; and b) asecond monomer comprising, from N- to C-terminal: i) a second variant Fcdomain protein; ii) an optional first domain linker; iii) a IL-12p40domain protein; iv) an optional second domain linker; v) a IL-12p35domain protein.

In some embodiments, the Fc-(scIL-12(p35/p40))₂ format provides ahomodimeric Fc fusion protein comprising: a) a first monomer comprising,from N- to C-terminal: i) a first variant Fc domain protein; ii) anoptional first domain linker; iii) a IL-12p35 domain protein; iv) anoptional second domain linker; v) a IL-12p40 domain protein; and b) asecond monomer comprising, from N- to C-terminal: i) a second variant Fcdomain protein; ii) an optional first domain linker; iii) a IL-12p35domain protein; iv) an optional second domain linker; v) a IL-12p40domain protein.

In some embodiments, the first domain linker and said second domainlinker have the same amino acid sequence. In some embodiments, the firstdomain linker and second domain linker have different amino acidsequences.

In some embodiments, the Fc variants comprise one or more skew, pI, andablation variants as provided herein. In one embodiment, Fc variantscomprise particular skew, pI, and ablation variants. In someembodiments, modifications promoting homodimerization of the first andthe second Fc domains are a set of amino acid substitutions selectedfrom the group consisting of L368D/K370S; S364K; S364K/E357L;S364K/E357Q; T411E/K360E/Q362E; D401K; T366S/L368A/Y407V; T366W;T366S/L368A/Y407V/Y349C; and T366W/S354C, according to EU numbering. Insome embodiments, the first and/or the second Fc domains have anadditional set of amino acid substitutions comprisingQ295E/N384D/Q418E/N421D, according to EU numbering. In some embodiments,the first and/or the second Fc domains have an additional set of aminoacid substitutions selected from the group consisting of G236R/L328R,E233P/L234V/L235A/G236_/S239K, E233P/L234V/L235A/G236_/S239K/A327G,E233P/L234V/L235A/G236_/S267K/A327G, E233P/L234V/L235A/G236_, andE233P/L234V/L235A/G236_/S267K, according to EU numbering. In someembodiments the first and second Fc domains further comprise amino acidsubstitutions M428L/N434S.

In this format, useful IL-12p40 protein domains include, but are notlimited to, SEQ ID NO: 4 (human IL-12 subunit beta (IL-12p40) matureform sequence). In this format, useful IL-12p40 variants include, butare not limited to, E59K, E59Q, D18N, D18K, E32Q, E33Q, D34N, D34K,Q42E, S43E, S43K, E45Q, Q56E, D62N, E73Q, D87N, K99E, K99Y, E100Q,N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D,N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E,N281D, N281Q, and E299Q. In this format, useful IL-12p40 variantsinclude, but are not limited to, N103D/N113D/N200D/N281D, Q42E/E45Q,E45Q/Q56E, Q42E/E59Q, Q56E/E59Q, Q42E/E45Q/Q56E, E45Q/Q56E/E59Q,E32Q/E59Q, D34N/E59K, D34N/E59K/K99E, D34K/E59K/K99E,E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q, E59Q/E187Q,S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E, E59Q/K260E,E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y, E59Y/K99Y,E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In this format, IL-12p35 protein domains include SEQ ID NO: 2 (humanIL-12 subunit alpha (IL-12p35) mature form sequence). In this format,useful IL-12p35 variants, include, but are not limited to, N21D, Q35D,E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A,M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K,E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q. Inthis format, useful IL-12p35 variants, include, but are not limited to,N71D/N85D/N195D, N151D/E153Q, N151D/D165N, Q130E/N151D, N151D/K158E,E79Q/N151D, D55Q/N151D, N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q,N71Q/N195Q, N85Q/N195Q, N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, andN85D/N195D.

In this format, useful embodiments include, but are not limited to,those found in FIGS. 14A-14B and FIG. 15 .

Potential embodiments of homodimeric Fc fusion proteins may use methodsand compositions described in the section above entitled “HeterodimericFc Fusion Proteins,” as appropriate with the goal of producing ahomodimer rather than heterodimer. Potential embodiments of homodimericFc fusion proteins may use any combination of methods and compositionsdescribed herein.

V. Interleukin 12

The present invention relates to the biologically functional form ofinterleukin 12. IL-12 heterodimeric Fc proteins comprising a IL-12p40variant and/or a IL-12p35 variants have reduced or decreased bindingaffinity to IL-12 receptors compared to a wild-type IL-12, including thep40/p35 heterodimer and the p40 homodimer. IL-12 heterodimeric Fcproteins of the present invention have reduced or decreased potencycompared to wild-type IL-12. IL-12 heterodimeric Fc proteins haveprolonged or increased serum half-life. IL-12 heterodimeric Fc proteinshave a reduced or decreased potential for toxicity. IL-12 heterodimericFc proteins of the present invention have reduced or decreased potencyin inducing STAT4 phosphorylation in immune cells such as, but notlimited to, T cells and NK cells. IL-12p40 variants comprise amino acidmodifications (e.g., substitutions, additions, and deletions) thatremove potential N-glycosylation sites. IL-12p35 variants comprise aminoacid modifications (e.g., substitutions, additions, and deletions) thatremove potential N-glycosylation sites. In some embodiments, the IL-12heterodimeric Fc proteins of the present invention has reduced/decreasedglycosylation compared to wild-type IL-12. In some embodiments, theglycosylation status of the IL-12 heterodimeric Fc proteins of thepresent invention is different than a wild-type IL-12 protein. IL-12heterodimeric Fc proteins of the present invention provide improvedpharmacokinetics compared to wild-type IL-12. Also, such IL-12heterodimeric Fc proteins are therapeutically effective and have animproved therapeutic index such as compared to wild-type IL-12. IL-12heterodimeric Fc proteins of the present invention can promote/induce Tcell activation. In some embodiments, administration of any one of theIL-12 heterodimeric Fc proteins to a patient induces T cell activationin the patient. In some instances, administration induces IFN-gammasecretion in the patient. In some cases, administration of the IL-12heterodimeric Fc proteins induces expansion of lymphocytes in thepatient. IL-12 heterodimeric Fc proteins are useful for treating cancersuch as solid tumor cancers or liquid tumor cancers. n some embodiments,administration of any one of the IL-12 heterodimeric Fc proteins to apatient, and in some cases, in combination with a checkpoint blockageagent reduces tumor size.

As stated above, the biologically functional form of interleukin 12 or“IL-12” is a heterodimer, composed of the IL-12p35 subunit (IL-12subunit alpha) and the IL-12p40 subunit (IL-12 subunit beta), hereondesignated as an “IL-12 heterodimeric complex”. This complex can be usedin two different formats. As shown in FIGS. 8A-B, the IL-12p40 subunitand the IL-12p35 subunits are not covalently attached to each other, butrather are covalently attached respectively to a first and a second Fcdomain which are assembled as a heterodimer. Alternatively, the IL-12p35and IL-12p40 subunits can be covalently attached, optionally using adomain linker (as described herein), as generally shown in FIGS. 8C-F,hereon designated a single-chain IL-12 complex or “scIL-12”. The orderof the two subunits in the scIL-12 may be designated as follows:“scIL-12(p40/p35)”, wherein the IL-12p40 subunit is N-terminally linked(with or without a domain linker) to the IL-12p35 subunit, or“scIL-12(p35/p40)”, wherein the IL-12p35 is N-terminally linked (with orwithout a domain linker) to the IL-12p40subunit.

In some embodiments, the human IL-12p35 protein has the amino acidsequence set forth in NCBI Ref. Seq. No. NP_000873.2 or SEQ ID NO:1(Human IL-12 subunit alpha (IL-12p35) precursor sequence as depicted inFIG. 1 ). In some cases, the coding sequence of human IL-12p35 is setforth in NCBI Ref. Seq. No. NM_000882.3. An exemplary IL-12p35 proteinof the Fc fusion heterodimeric protein outlined herein can have theamino acid sequence of SEQ ID NO:2 (Human IL-12 subunit alpha (IL-12p35)mature form sequence as depicted in FIG. 1 ) or amino acids 23-219 ofSEQ ID NO:1. In some embodiments, the IL-12p35 protein has at least 90%,e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to SEQ ID NO:2. The IL-12p35 protein of the Fc fusion proteincan have 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid mutations.

In some embodiments, the human IL-12p40 protein has the amino acidsequence set forth in NCBI Ref. Seq. No. NP_002178.3 or SEQ ID NO:3(Human IL-12 subunit beta (IL-12p40) precursor sequence as depicted inFIG. 1 ). In some cases, the coding sequence of human IL-12p40 is setforth in NCBI Ref. Seq. No. NM_002187.3. An exemplary IL-12p40 proteinof the Fc fusion protein outlined herein can have the amino acidsequence of SEQ ID NO:4 (Human IL-12 subunit beta (IL-12p40) mature formsequence as depicted in FIG. 1 ) or amino acids 23-328 of SEQ ID NO:3.In some embodiments, the IL-12p40 protein has at least 90%, e.g., 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identityto SEQ ID NO:4. The IL-12p40 protein of the Fc fusion protein can have1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid mutations.

The present invention also provides variant IL-12p40 subunits andvariant IL-12p35 subunits. These variants find use as part of thebiologically functional IL-12 complex as well as any of the IL-12-Fcfusions described herein.

A. Expression Variants

As a preliminary matter, the IL-12p40 and IL-12p35 subunits of theinvention also include variants to remove potential N-glycosylationsites designed to reduce heterogeneity.

Such potential N-glycosylation sites on IL-12p40 at which amino acidmodifications can be introduced include N103, N113, N200, and N281(numbered according to the Human IL-12 subunit beta (IL-12p40 matureform sequence as depicted in FIG. 1 ). Illustrative modifications at oneor more of these sites may be selected from the group consisting of:N103D, N103Q, N113D, N113Q, N200D, N200Q, N281D, and N281Q. IL-12p40expression variants can include one or more modifications at thesesites. Accordingly, in one embodiment, the IL-12p40 variant comprisesN103D/N113D/N200D/N281D. In one embodiment, the IL-12p40 variantcomprises N103D/N113D. In one embodiment, the IL-12p40 variant comprisesN103D/N200D. In one embodiment, the IL-12p40 variant comprisesN103D/N281D. In one embodiment, the IL-12p40 variant comprisesN113D/N200D. In one embodiment, the IL-12p40 variant comprisesN113D/N281D. In one embodiment, the IL-12p40 variant comprisesN200D/N281D. In one embodiment, the IL-12p40 variant comprisesN103D/N113D/N200D. In one embodiment, the IL-12p40 variant comprisesN103D/N113D/N281D. In one embodiment, the IL-12p40 variant comprisesN/103D/N200D/N281D. In one embodiment, the IL-12p40 variant comprisesN113D/N200D/N281D. In one embodiment, the IL-12p40 variant comprisesN103Q/N113Q. In one embodiment, the IL-12p40 variant comprisesN103Q/N200Q. In one embodiment, the IL-12p40 variant comprisesN103Q/N281Q. In one embodiment, the IL-12p40 variant comprisesN113Q/N200Q. In one embodiment, the IL-12p40 variant comprisesN113Q/N281Q. In one embodiment, the IL-12p40 variant comprisesN103Q/N113Q/N200Q. In one embodiment, the IL-12p40 variant comprisesN103Q/N113Q/N281Q. In one embodiment, the IL-12p40 variant comprisesN103Q/N200Q/N281Q. In one embodiment, the IL-12p40 variant comprisesN113Q/N200Q/N281Q. These modifications can be used alone or incombination with any other IL-12p40 variants, such as affinity variants.

Such potential N-glycosylation sites on IL-12p35 at which amino acidmodifications can be introduced at one or more of the sites selectedfrom the group consisting of: N71, N85, and N195 (numbered according tothe Human IL-12 subunit alpha (IL-12p35) mature form sequence asdepicted in FIG. 1 ). Illustrative modifications at these sites includeN71D, N71Q, N85D, N85Q, N195D, and N195Q. IL-12p35 variants can includeone or more modifications at these sites. Accordingly, in oneembodiment, the IL-12p35 variant comprises N71D/N85D/N195D. Thesemodifications can be used alone or in combination with any otherIL-12p35 variants, such as affinity variants.

The IL-12p40 subunit has a free cysteine at position 252 (numberedaccording to the Human IL-12 subunit beta (IL-12p40) mature formsequence as depicted in FIG. 1 ) which may bond with other freecysteines leading at least to heterogeneity and at worse toimmunogenicity. Accordingly, IL-12p40 variants were engineered to removethe free cysteine, for example, by introducing C252S modification(although other substitutions may also be used). Modification of C252(e.g. C252S) can be used alone or in combination with any other IL-12p40variants, such as affinity or expression variants. Illustrative IL-12p40variants comprising a modification at C252 to remove the free cysteineare depicted in FIG. 65 . Illustrative IL-12-Fc fusions proteins weregenerated with the additional variant IL-12p40 subunits, sequences forwhich are depicted in FIG. 66 , and produced as generally described inExample 1B. These modifications can be used alone or in combination withany other IL-12p40 variants, such as affinity variants.

B. Affinity and Potency Variants

The invention provides IL-12p40 variants and IL-12p35 variants whichform biologically functional IL-12 with altered, that is either reducedor increased, binding affinity for IL-12 receptors. In some cases, thevariant IL-12p40 subunit has altered, that is either reduced orincreased, binding affinity for IL-12 receptor subunit beta-1(IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2), and/or the IL-12receptor complex. In some cases, the variant IL-12p35 has altered, thatis either reduced or increased, binding affinity for IL-12 receptorsubunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2 (IL-12Rβ2),and/or the IL-12 receptor complex. The invention also provides IL-12p40variants and IL-12p35 variants which form biologically functional IL-12with altered, that is either reduced or increased, potency compared towild-type IL-12p40 and IL-12p35.

Suitable sites on IL-12p40 at which amino acid modifications can be beintroduced are selected from the group consisting of: E3, D7, E12, D14,W15, P17, D18, A19, P20, G21, E22, M23, D29, E32, E33, D34, L40, D41,Q42, S43, E45, L47, T54, 155, Q56, K58, E59, F60, G61, D62, Q65, Y66,E73, K84, E86, D87, G88, 189, W90, D93, D97, K99, E100, K102, N103,K104, F106, E110, N113, Y114, D129, D142, Q144, E156, R159, D161, N162,K163, D166, D170, Q172, D174, A176, C177, P178, A179, A180, E181, S183,P185, E187, N200, S204, F206, R208, D209, D214, N218, Q220, N226, Q229,E231, E235, T242, P243, S245, Y246, F247, S248, C252, Q256, K258, K260,E262, K264, D265, D270, N281, Q289, D290, R291, Y292, Y293, and E299(numbered according to the Human IL-12 subunit beta (IL-12p40 matureform sequence as depicted in FIG. 1 ). Illustrative modifications atthese sites can be selected from the group consisting of: D18N, D18K,E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N,E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N,R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N,K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q. IL-12p40 affinityvariants can include modifications at one or more of these sites.Accordingly, in one embodiment, the IL-12p40 variant comprisesN103D/N113D/N200D/N281D. In another embodiment, the IL-12p40 variantcomprises Q42E/E45Q. In another embodiment, the IL-12p40 variantcomprises E45Q/Q56E. In another embodiment, the IL-12p40 variantcomprises Q42E/E59Q. In another embodiment, the IL-12p40 variantcomprises Q56E/E59Q. In another embodiment, the IL-12p40 variantcomprises Q42E/E45Q/Q56E. In another embodiment, the IL-12p40 variantcomprises E45Q/Q56E/E59Q. In another embodiment, the IL-12p40 variantcomprises E32Q/E59Q. In another embodiment, the IL-12p40 variantcomprises D34N/E59K. In another embodiment, the IL-12p40 variantcomprises D34N/E59K/K99E. In another embodiment, the IL-12p40 variantcomprises D34K/E59K/K99E. In another embodiment, the IL-12p40 variantcomprises E32Q/D34N/E59K/K99E. In another embodiment, the IL-12p40variant comprises E32K/D34N/E59K/K99E. In another embodiment, theIL-12p40 variant comprises D34N/E59Q. In another embodiment, theIL-12p40 variant comprises E59Q/E187Q. In another embodiment, theIL-12p40 variant comprises S43E/E59Q. In another embodiment, theIL-12p40 variant comprises S43K/E49Q. In another embodiment, theIL-12p40 variant comprises E59Q/K163E. In another embodiment, theIL-12p40 variant comprises E59Q/K99E. In another embodiment, theIL-12p40 variant comprises E59Q/K258E. In another embodiment, theIL-12p40 variant comprises E59Q/K260E. In another embodiment, theIL-12p40 variant comprises E59K/K99E. In another embodiment, theIL-12p40 variant comprises D18K/E59K/K99E. In another embodiment, theIL-12p40 variant comprises E59K/K99E/K264E. In another embodiment, theIL-12p40 variant comprises E59K/K99Y. In another embodiment, theIL-12p40 variant comprises E59Y/K99Y. In another embodiment, theIL-12p40 variant comprises E59Y/K99E. In another embodiment, theIL-12p40 variant comprises E45K/E59K/K99E. In another embodiment, theIL-12p40 variant comprises E59K/K99E/Q144E. In another embodiment, theIL-12p40 variant comprises E59K/K99E/Q144K. In another embodiment, theIL-12p40 variant comprises E59K/K99E/R159E. In another embodiment, theIL-12p40 variant comprises E59K/K99E/K264E. In another embodiment, theIL-12p40 variant comprises D18K/E59K/K99E/K264E. In another embodiment,the IL-12p40 variant comprises DI8K/E59K/K99E/C252S. In anotherembodiment, the IL-12p40 variant comprises D18K/E59K/K99E/C252S/K264E.In another embodiment, the IL-12p40 variant comprises E59K/K99Y/C252S.In another embodiment, the IL-12p40 variant comprisesE59K/K99E/C252S/K264E. In another embodiment, the IL-12p40 variantcomprises E59K/K99E/C252S. In another embodiment, the IL-12p40 variantcomprises N103D/N113D. In another embodiment, the IL-12p40 variantcomprises N103D/N200D. In another embodiment, the IL-12p40 variantcomprises N103D/N281D. In another embodiment, the IL-12p40 variantcomprises N113D/N200D. In another embodiment, the IL-12p40 variantcomprises N113D/N281D. In another embodiment, the IL-12p40 variantcomprises N200D/N281D. In another embodiment, the IL-12p40 variantcomprises N103D/N113D/N200D. In another embodiment, the IL-12p40 variantcomprises N103D/N113D/N281D. In another embodiment, the IL-12p40 variantcomprises N103D/N200D/N281D. In another embodiment, the IL-12p40 variantcomprises N113D/N200D/N281D. In another embodiment, the IL-12p40 variantcomprises N103Q/N113Q. In another embodiment, the IL-12p40 variantcomprises N103Q/N200Q. In another embodiment, the IL-12p40 variantcomprises N103Q/N281Q. In another embodiment, the IL-12p40 variantcomprises N113Q/N200Q. In another embodiment, the IL-12p40 variantcomprises N113Q/N281Q. In another embodiment, the IL-12p40 variantcomprises N200Q/N281Q. In another embodiment, the IL-12p40 variantcomprises N103Q/N113Q/N200Q. In another embodiment, the IL-12p40 variantcomprises N103Q/N113Q/N281Q. In another embodiment, the IL-12p40 variantcomprises N103Q/N200Q/N281Q. In another embodiment, the IL-12p40 variantcomprises N113Q/N200Q/N281Q. In another embodiment, the IL-12p40 variantcomprises N103Q/N113Q/N200Q/N281Q. In another embodiment, the IL-12p40variant comprises E59K/K99E/N103Q/C252S/K264E. In another embodiment,the IL-12p40 variant comprises E59K/K99E/N113Q/C252S/K264E. In anotherembodiment, the IL-12p40 variant comprises E59K/K99E/N200Q/C252S/K264E.In another embodiment, the IL-12p40 variant comprisesE59K/K99E/N281Q/C252S/K264E. In another embodiment, the IL-12p40 variantcomprises E59K/K99E/N103Q/N113Q/C252S/K264E. In another embodiment, theIL-12p40 variant comprises E59K/K99E/N103Q/N200Q/C252S/K264E. In anotherembodiment, the IL-12p40 variant comprisesE59K/K99E/N103Q/N281Q/C252S/K264E. In another embodiment, the IL-12p40variant comprises E59K/K99E/N113Q/N200Q/C252S/K264E. In anotherembodiment, the IL-12p40 variant comprisesE59K/K99E/N113Q/N281Q/C252S/K264E. In another embodiment, the IL-12p40variant comprises E59K/K99E/N200Q/N281Q/C252S/K264E. In anotherembodiment, the IL-12p40 variant comprisesE59K/K99E/N103Q/N113Q/N200Q/C252S/K264E. In another embodiment, theIL-12p40 variant comprises E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E. Inanother embodiment, the IL-12p40 variant comprisesE59K/K99E/N113Q/N200Q/N281Q/C252S/K264E. In another embodiment, theIL-12p40 variant comprisesE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

Additionally, these modifications can be used alone or in combinationwith any other IL-12p40 variants, such as expression variants.

The IL-12p40 subunit has a free cysteine at position 252 (numberedaccording to the Human IL-12 subunit beta (IL-12p40) mature formsequence as depicted in FIG. 1 ) which may bond with other freecysteines leading at least to heterogeneity and at worse toimmunogenicity. Accordingly, IL-12p40 variants were engineered to removethe free cysteine, for example, by introducing C252S modification(although other substitutions may also be used). Modification of C252(e.g. C252S) can be used alone or in combination with any other IL-12p40variants, such as affinity or expression variants. Illustrative IL-12p40variants comprising a modification at C252 to remove the free cysteineare depicted in FIG. 65 . Illustrative IL-12-Fc fusions proteins weregenerated with the additional variant IL-12p40 subunits, sequences forwhich are depicted in FIG. 66 , and produced as generally described inExample 1B. Additionally, these modifications can be used alone or incombination with any other IL-12p40 variants, such as expressionvariants.

Suitable sites on IL-12p35 at which amino acid modifications can beintroduced are selected from the group consisting of: Q20, N21, Q35,E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63, L64, P65, E67,L68, N71, S73, C74, L75, N76, E79, N85, F96, M97, L89, L124, M125, Q130,Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171, R181,1182, R183, V185, T186, D188, R189, V190, S192, Y193, and A196 (numberedaccording to the Human IL-12 subunit alpha (IL-12p35) mature formsequence as depicted in FIG. 1 ). Illustrative modifications areselected from the group consisting of: N21D, Q35D, E38Q, D55Q, D55K,N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A, L124A,M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q,K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q. IL-12p35 affinityvariants can include modifications at one or more of these sites.Accordingly, in one embodiment, the IL-12p35 variant comprisesN71D/N85D/N195D. In another embodiment, the IL-12p35 variant comprisesN151D/E153Q. In another embodiment, the IL-12p35 variant comprisesN151D/D165N. In another embodiment, the IL-12p35 variant comprisesQ130E/N151D. In another embodiment, the IL-12p35 variant comprisesN151D/K158E. In another embodiment, the IL-12p35 variant comprisesE79Q/N151D. In another embodiment, the IL-12p35 variant comprisesD55Q/N151D. In another embodiment, the IL-12p35 variant comprisesN136D/N151D. In another embodiment, the IL-12p35 variant comprisesN21D/N151D. In another embodiment, the IL-12p35 variant comprisesE143Q/N151D. In another embodiment, the IL-12p35 variant comprisesN71Q/N85Q. In another embodiment, the IL-12p35 variant comprisesN71Q/N195Q. In another embodiment, the IL-12p35 variant comprisesN85Q/N195Q. In another embodiment, the IL-12p35 variant comprisesN71Q/N85Q/N195Q. In another embodiment, the IL-12p35 variant comprisesN71D/N85D. In another embodiment, the IL-12p35 variant comprisesN71D/N195D. In another embodiment, the IL-12p35 variant comprisesN85D/N195D.

Additionally, these modifications can be used alone or in combinationwith any other IL-12p35 variants, such as expression variants.

A biologically functional IL-12 heterodimeric complex can comprise awild-type IL-12p40 subunit and a wild-type IL-12p35 subunit, a variantIL-12p40 subunit and a wild-type IL-12p35 subunit, a wild-type IL-12p40subunit and a variant IL-12p35 subunit, or a variant IL-12p40 subunitand a variant IL-12p35 subunit.

A biologically functional IL-12 bivalent homodimeric complex cancomprise a wild-type IL-12p40 subunit and a wild-type IL-12p35 subunit,a variant IL-12p40 subunit and a wild-type IL-12p35 subunit, a wild-typeIL-12p40 subunit and a variant IL-12p35 subunit, or a variant IL-12p40subunit and a variant IL-12p35 subunit.

VI. Domain Linkers

In some embodiments, the IL-12p35 and IL-12p40 subunits are attachedtogether via a linker. Optionally, the subunits are not attached via alinker. In other embodiments, the IL-12p35 and IL-12p40 subunits arenoncovalently attached. In some embodiments, the IL-12p35 subunit isattached to an Fc domain via a linker. In certain embodiments, theIL-12p35 subunit is attached to an Fc domain directly, such as without alinker. In other embodiments, the IL-12p40 subunit is attached to an Fcdomain via a linker. In other embodiments, the IL-12p40 subunit isattached to an Fc domain directly. In some cases, a linker is not usedto attach the IL-12p35 subunit or IL-12p40 subunit to an Fc domain.

In some embodiments, the linker is a “domain linker”, used to link anytwo domains as outlined herein together. While any suitable linker canbe used, many embodiments utilize a glycine-serine polymer, includingfor example (GS)_(n) (SEQ ID NO: 463), (GSGGS)_(n)(SEQ ID NO: 464),(GGGGS)_(n) (SEQ ID NO: 465), and (GGGS)_(n) (SEQ ID NO: 432), where nis an integer of at least 0 (and generally from 0 to 1 to 2 to 3 to 4 to5), as well as any peptide sequence that allows for recombinantattachment of the two domains with sufficient length and flexibility toallow each domain to retain its biological function. In certain cases,useful linkers include (GGGGS)₀ (“GGGGS” disclosed as SEQ ID NO: 9) or(GGGGS)₁ (SEQ ID NO: 9) or (GGGGS)₂(SEQ ID NO: 10). Illustrative domainlinkers are depicted in FIG. 6 . In some cases, and with attention beingpaid to “strandedness”, as outlined below, charged domain linkers can beused as discussed herein.

In addition, it has been previously reported that the serine in Gly-Serlinkers in Fc fusions may be subject to O-glycosylation. Accordingly, afinal approach to decrease heterogeneity (in the context of Fc fusions)is to replace the Gly-Ser linkers used thus far with Gly-Ala linkers.

VII. Useful Formats of the Invention

As shown in FIGS. 8A-F there are a number of useful formats of theheterodimeric fusion proteins of the invention. In general, theheterodimeric fusion proteins of the invention have two functionalcomponents: an IL-12 heterodimer component and an Fc component, both ofwhich can take different forms as outlined herein and both of which canbe combined with the other component in any configuration.

In some embodiments, the IL-12p35 and IL-12p40 subunits are covalentlylinked, optionally with a domain linker, and is referred to herein as asingle-chain IL-12 complex or “scIL-12”. The scIL-12 can comprise eitherIL-12p35 N-terminally linked to IL-12p40 or IL-12p40 N-terminally linkedto IL-12p35, optionally with a domain linker. The order of the twosubunits in the scIL-12 may be designated as follows:“scIL-12(p40/p35)”, wherein the IL-12p40 subunit is N-terminally linked(with or without a domain linker) to the IL-12p35 subunit, or“scIL-12(p35/p40)”, wherein the IL-12p35 is N-terminally linked (with orwithout a domain linker) to the IL-12p340subunit.

In some embodiments, the IL-12p35 and IL-12p40 subunits are notcovalently linked, but rather are covalently attached respectively to afirst and a second Fc domain which are assembled as a heterodimer.

The first and the second Fc domains can have a set of amino acidsubstitutions selected from the group consisting of a) L368D/K370S andS364K; b) L368D/K370S and S364K/E357L; c) L368D/K370S and S364K/E357Q;d) S267K/L368D/K370S; e) T411E/K360E/Q362E and D401K; f) L368E/K370S andS364K; g) K370S and S364K/E357Q; and h) T366S/L368A/Y407V and T366W(optionally including a bridging disulfide, T366S/L368A/Y407V/Y349C andT366W/S354C), according to EU numbering.

In some embodiments, the first and/or the second Fc domains have anadditional set of amino acid substitutions comprisingQ295E/N384D/Q418E/N421D, according to EU numbering.

Optionally, the first and/or the second Fc domains have an additionalset of amino acid substitutions selected from the group consisting ofG236R/L328R, E233P/L234V/L235A/G236_/S239K,E233P/L234V/L235A/G236_/S239K/A327G,E233P/L234V/L235A/G236_/S267K/A327G, E233P/L234V/L235A/G236_, andE233P/L234V/L235A/G236_/S267K, according to EU numbering.

Optionally, the first and/or second Fc domains have 428L/434S variantsfor half life extension.

A. IL-12-heteroFc Format

In one embodiment, the present invention provides the N-terminal IL-12heterodimeric Fc fusion or “IL-12-heteroFc” format. In this embodiment,as shown in FIG. 8A, the heterodimeric fusion protein comprises twomonomers. The first monomer comprises (from N- to C-terminus)IL-12p40-optional domain linker-Fc. The second monomer comprises (fromN- to C-terminus) the IL-12p35-optional domain linker-Fc.

In the IL-12-heteroFc format, a preferred embodiment utilizes the skewvariant pair S364K/E357Q:L368D/K370S.

In the IL-12-heteroFc format, a preferred embodiment is shown in FIG. 9(XENP27201 including chain 1 chain 2).

In some embodiments, the IL-12-heteroFc format provides a heterodimericFc fusion protein comprising: a) a first monomer comprising, from N- toC-terminal: i) a IL-12p40 domain protein; ii) an optional first domainlinker; iii) a first variant Fc domain protein; and b) a second monomercomprising, from N- to C-terminal: i) a IL-12p35 domain protein; ii)optionally a second domain linker; iii) a second variant Fc domainprotein.

In some embodiments, the Fc variants comprise one or more skew, pI, andablation variants as provided herein. In one embodiment, Fc variantscomprise particular skew, pI, and ablation variants. In someembodiments, modifications promoting heterodimerization of the first andthe second Fc domains are a set of amino acid substitutions selectedfrom the group consisting of L368D/K370S and S364K; L368D/K370S andS364K/E357L; L368D/K370S and S364K/E357Q; T411E/K360E/Q362E and D401K;L368E/K370S and S364K; K370S and S364K/E357Q; T366S/L368A/Y407V andT366W; T366S/L368A/Y407V/Y349C and T366W/S354C, according to EUnumbering. In some embodiments, the first and/or the second Fc domainshave an additional set of amino acid substitutions comprisingQ295E/N384D/Q418E/N421D, according to EU numbering. In some embodiments,the first and/or the second Fc domains have an additional set of aminoacid substitutions selected from the group consisting of G236R/L328R,E233P/L234V/L235A/G236_/S239K, E233P/L234V/L235A/G236_/S239K/A327G,E233P/L234V/L235A/G236_/S267K/A327G, E233P/L234V/L235A/G236_, andE233P/L234V/L235A/G236_/S267K, according to EU numbering. In someembodiments the first and second Fc domains further comprise amino acidsubstitutions M428L/N434S.

In this format, useful IL-12p40 protein domains include, but are notlimited to, SEQ ID NO: 4 (human IL-12 subunit beta (IL-12p40) matureform sequence). In this format, useful IL-12p40 variants include, butare not limited to, E59K, E59Q, D18N, D18K, E32Q, E33Q, D34N, D34K,Q42E, S43E, S43K, E45Q, Q56E, D62N, E73Q, D87N, K99E, K99Y, E100Q,N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D,N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E,N281D, N281Q, and E299Q. In this format, useful IL-12p40 variantsinclude, but are not limited to, N103D/N113D/N200D/N281D, Q42E/E45Q,E45Q/Q56E, Q42E/E59Q, Q56E/E59Q, Q42E/E45Q/Q56E, E45Q/Q56E/E59Q,E32Q/E59Q, D34N/E59K, D34N/E59K/K99E, D34K/E59K/K99E,E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q, E59Q/E187Q,S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E, E59Q/K260E,E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y, E59Y/K99Y,E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In this format, IL-12p35 protein domains include SEQ ID NO: 2 (humanIL-12 subunit alpha (IL-12p35) mature form sequence). In this format,useful IL-12p35 variants, include, but are not limited to, N21D, Q35D,E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A,M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K,E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q. Inthis format, useful IL-12p35 variants, include, but are not limited to,N71D/N85D/N195D, N151D/E153Q, N151D/D165N, Q130E/N151D, N151D/K158E,E79Q/N151D, D55Q/N151D, N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q,N71Q/N195Q, N85Q/N195Q, N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, andN85D/N195D.

In this format, useful combinations of a variant IL-12p40 subunit andIL-12p35 subunit include, but are not limited to IL-12p40(E59K/K99Y) andIL-12p35(SEQ ID NO: 2), IL-12p40(D18K/E59K/K99E) and IL-12p35 (SEQ IDNO: 2), IL-12p40(E59K/K99E/K264E) and IL-12p35(SEQ ID NO: 2),IL-12p40(D18K/E59K/K99E/K264E) and IL-12p35(SEQ ID NO: 2),IL-12p40(D18K/E59K/K99E/C252S) and IL-12p35(SEQ ID NO: 2),IL-12p40(D18K/E59K/K99E/C252S/K264E) and IL-12p35(SEQ ID NO: 2),IL-12p40(E59K/K99Y/C252S) and IL-12p35(SEQ ID NO: 2),IL-12p40(E59K/K99E/C252S/K264E) and IL-12p35(SEQ ID NO: 2), andIL-12p40(E59K/K99E/C252S) and IL-12p35(SEQ ID NO: 2).

In this format, useful combinations of a variant IL-12p40 subunit and aIL-12p35 subunit include, but are not limited to a combination of anIL-12p35 comprising SEQ ID NO: 2 (human IL-12 subunit alpha (IL-12p35)mature form sequence) and a variantIL-12p40 comprising one or moresubstitutions including, but not limited, to E59K, E59Q, D18N, D18K,E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, D62N, E73Q, D87N,K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E,K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E,K260E, E262Q, K264E, N281D, N281Q, E299Q, N103D/N113D/N200D/N281D,Q42E/E45Q, E45Q/Q56E, Q42E/E59Q, Q56E/E59Q, Q42E/E45Q/Q56E,E45Q/Q56E/E59Q, E32Q/E59Q, D34N/E59K, D34N/E59K/K99E, D34K/E59K/K99E,E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q, E59Q/E187Q,S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E, E59Q/K260E,E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y, E59Y/K99Y,E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In this format, useful combinations of IL-12p40 and IL-12p35 variantsinclude, but are not limited to a combination of an IL-12p40 comprisingSEQ ID NO: 4 (human IL-12 subunit beta (IL-12p40) mature form sequence)and an IL-12p35 comprising N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q,L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A, L124A, M125A, Q130E,Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q,E163Q, D165N, I171A, N195D, N195Q, N71D/N85D/N195D, N151D/E153Q,N151D/D165N, Q130E/N151D, N151D/K158E, E79Q/N151D, D55Q/N151D,N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q, N71Q/N195Q, N85Q/N195Q,N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, and N85D/N195D.

In this format, useful combinations of IL-12p40 variants and IL-12p35variants include, but are not limited to, a combination of an IL-12p40variant comprising one or more amino acid substitution selected from thegroup consisting of: E59K, E59Q, D18N, D18K, E32Q, E33Q, D34N, D34K,Q42E, S43E, S43K, E45Q, Q56E, D62N, E73Q, D87N, K99E, K99Y, E100Q,N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D,N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E,N281D, N281Q, E299Q, N103D/N113D/N200D/N281D, Q42E/E45Q, E45Q/Q56E,Q42E/E59Q, Q56E/E59Q, Q42E/E45Q/Q56E, E45Q/Q56E/E59Q, E32Q/E59Q,D34N/E59K, D34N/E59K/K99E, D34K/E59K/K99E, E32Q/D34N/E59K/K99E,E32K/D34N/E59K/K99E, D34N/E59Q, E59Q/E187Q, S43E/E59Q, S43K/E49Q,E59Q/K163E, E59Q/K99E, E59Q/K258E, E59Q/K260E, E59K/K99E,D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y, E59Y/K99Y, E59Y/K99E,E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K, E59K/K99E/R159E,E59K/K99E/K264E, D18K/E59K/K99E/K264E, DI8K/E59K/K99E/C252S,D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S, E59K/K99E/C252S/K264E,E59K/K99E/C252S, N103D/N113D, N103D/N200D, N103D/N281D, N113D/N200D,N113D/N281D, N200D/N281D, N103D/N113D/N200D, N103D/N113D/N281D,N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q, N103Q/N200Q,N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q, N103Q/N113Q/N200Q,N103Q/N113Q/N281Q, N103Q/N200Q/N281Q, N113Q/N200Q/N281Q,N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E; and an IL-12p35 variantcomprising one or more substitutions selected from the group consistingof: N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D,N85Q, L89A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E,N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D,N195Q, N71D/N85D/N195D, N151D/E153Q, N151D/D165N, Q130E/N151D,N151D/K158E, E79Q/N151D, D55Q/N151D, N136D/N151D, N21D/N151D,E143Q/N151D, N71Q/N85Q, N71Q/N195Q, N85Q/N195Q, N71Q/N85Q/N195Q,N71D/N85D, N71D/N195D, and N85D/N195D.

In this format, useful embodiments include but are not limited to thosein which a variant IL-12p40 subunit domain is attached to said first Fcdomain using a first domain linker and/or said IL-12p35 subunit domainis attached to said second Fc domain using a second domain linker.

In this format, useful embodiments include but are not limited toXENP31251, XENP31254, XENP31258, XENP32186, XENP32187, XENP32188,XENP32189, XENP32190, and XENP32191. In further embodiments, XENP31251,XENP31254, XENP31258, XENP32186, XENP32187, XENP32188, XENP32189,XENP32190, and XENP32191 include further glycoengineering.

In this format, useful embodiments include, but are not limited to,those found in FIGS. 44A-44K, and FIGS. 66A-66Q.

B. heteroFc-IL-12 Format

In another embodiment, the present invention provides the C-terminalIL-12 heterodimeric Fc fusion or “heteroFc-IL-12” format. In thisembodiment, as shown in FIG. 8B, the heterodimeric fusion proteincomprises two monomers. The first monomer comprises (from N-toC-terminus) Fc-optional domain linker-IL-12p40. The second monomercomprises (from N-to C-terminus) Fc-optional domain linker-IL-12p35.

In the heteroFc-IL-12 format, a preferred embodiment utilizes the skewvariant pair S364K/E357Q:L368D/K370S.

In the IL-12-heteroFc format, a preferred embodiment is shown in FIG. 10(XENP27202 including chain 1 chain 2).

In some embodiments, the heteroFc-IL-12 format provides a heterodimericFc fusion protein comprising: a) a first monomer comprising, from N- toC-terminal: i) a first variant Fc domain protein; ii) an optional firstdomain linker; iii) a IL-12p40 domain protein; and b) a second monomercomprising, from N- to C-terminal: i) a second variant Fc domainprotein; ii) optionally a second domain linker; iii) a IL-12p35 domainprotein.

In some embodiments, the Fc variants comprise one or more skew, pI, andablation variants as provided herein. In one embodiment, Fc variantscomprise particular skew, pI, and ablation variants. In someembodiments, modifications promoting heterodimerization of the first andthe second Fc domains are a set of amino acid substitutions selectedfrom the group consisting of L368D/K370S and S364K; L368D/K370S andS364K/E357L; L368D/K370S and S364K/E357Q; T411E/K360E/Q362E and D401K;L368E/K370S and S364K; K370S and S364K/E357Q; T366S/L368A/Y407V andT366W; T366S/L368A/Y407V/Y349C and T366W/S354C, according to EUnumbering. In some embodiments, the first and/or the second Fc domainshave an additional set of amino acid substitutions comprisingQ295E/N384D/Q418E/N421D, according to EU numbering. In some embodiments,the first and/or the second Fc domains have an additional set of aminoacid substitutions selected from the group consisting of G236R/L328R,E233P/L234V/L235A/G236_/S239K, E233P/L234V/L235A/G236_/S239K/A327G,E233P/L234V/L235A/G236_/S267K/A327G, E233P/L234V/L235A/G236_, andE233P/L234V/L235A/G236_/S267K, according to EU numbering. In someembodiments the first and second Fc domains further comprise amino acidsubstitutions M428L/N434S.

In this format, useful IL-12p40 protein domains include, but are notlimited to, SEQ ID NO: 4 (human IL-12 subunit beta (IL-12p40) matureform sequence). In this format, useful IL-12p40 variants include, butare not limited to, E59K, E59Q, D18N, D18K, E32Q, E33Q, D34N, D34K,Q42E, S43E, S43K, E45Q, Q56E, D62N, E73Q, D87N, K99E, K99Y, E100Q,N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D,N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E,N281D, N281Q, and E299Q. In this format, useful IL-12p40 variantsinclude, but are not limited to, N103D/N113D/N200D/N281D, Q42E/E45Q,E45Q/Q56E, Q42E/E59Q, Q56E/E59Q, Q42E/E45Q/Q56E, E45Q/Q56E/E59Q,E32Q/E59Q, D34N/E59K, D34N/E59K/K99E, D34K/E59K/K99E,E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q, E59Q/E187Q,S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E, E59Q/K260E,E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y, E59Y/K99Y,E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In this format, IL-12p35 protein domains include SEQ ID NO: 2 (humanIL-12 subunit alpha (IL-12p35) mature form sequence). In this format,useful IL-12p35 variants, include, but are not limited to, N21D, Q35D,E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A,M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K,E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q. Inthis format, useful IL-12p35 variants, include, but are not limited to,N71D/N85D/N195D, N151D/E153Q, N151D/D165N, Q130E/N151D, N151D/K158E,E79Q/N151D, D55Q/N151D, N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q,N71Q/N195Q, N85Q/N195Q, N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, andN85D/N195D.

In this format, useful combinations of a variant IL-12p40 subunit andIL-12p35 subunit include, but are not limited to IL-12p40(E59K/K99Y) andIL-12p35(SEQ ID NO: 2), IL-12p40(D18K/E59K/K99E) and IL-12p35 (SEQ IDNO: 2), IL-12p40(E59K/K99E/K264E) and IL-12p35(SEQ ID NO: 2),IL-12p40(D18K/E59K/K99E/K264E) and IL-12p35(SEQ ID NO: 2),IL-12p40(D18K/E59K/K99E/C252S) and IL-12p35(SEQ ID NO: 2),IL-12p40(D18K/E59K/K99E/C252S/K264E) and IL-12p35(SEQ ID NO: 2),IL-12p40(E59K/K99Y/C252S) and IL-12p35(SEQ ID NO: 2),IL-12p40(E59K/K99E/C252S/K264E) and IL-12p35(SEQ ID NO: 2), andIL-12p40(E59K/K99E/C252S) and IL-12p35(SEQ ID NO: 2).

In this format, useful combinations of a variant IL-12p40 subunit and aIL-12p35 subunit include, but are not limited to a combination of anIL-12p35 comprising SEQ ID NO: 2 (human IL-12 subunit alpha (IL-12p35)mature form sequence) and a variantIL-12p40 comprising one or moresubstitutions including, but not limited, to E59K, E59Q, D18N, D18K,E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, D62N, E73Q, D87N,K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E,K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E,K260E, E262Q, K264E, N281D, N281Q, E299Q, N103D/N113D/N200D/N281D,Q42E/E45Q, E45Q/Q56E, Q42E/E59Q, Q56E/E59Q, Q42E/E45Q/Q56E,E45Q/Q56E/E59Q, E32Q/E59Q, D34N/E59K, D34N/E59K/K99E, D34K/E59K/K99E,E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q, E59Q/E187Q,S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E, E59Q/K260E,E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y, E59Y/K99Y,E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In this format, useful combinations of IL-12p40 and IL-12p35 variantsinclude, but are not limited to a combination of an IL-12p40 comprisingSEQ ID NO: 4 (human IL-12 subunit beta (IL-12p40) mature form sequence)and an IL-12p35 comprising N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q,L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A, L124A, M125A, Q130E,Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q,E163Q, D165N, I171A, N195D, N195Q, N71D/N85D/N195D, N151D/E153Q,N151D/D165N, Q130E/N151D, N151D/K158E, E79Q/N151D, D55Q/N151D,N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q, N71Q/N195Q, N85Q/N195Q,N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, and N85D/N195D.

In this format, useful combinations of IL-12p40 variants and IL-12p35variants include, but are not limited to, a combination of an IL-12p40variant comprising one or more amino acid substitution selected from thegroup consisting of: E59K, E59Q, D18N, D18K, E32Q, E33Q, D34N, D34K,Q42E, S43E, S43K, E45Q, Q56E, D62N, E73Q, D87N, K99E, K99Y, E100Q,N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D,N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E,N281D, N281Q, E299Q, N103D/N113D/N200D/N281D, Q42E/E45Q, E45Q/Q56E,Q42E/E59Q, Q56E/E59Q, Q42E/E45Q/Q56E, E45Q/Q56E/E59Q, E32Q/E59Q,D34N/E59K, D34N/E59K/K99E, D34K/E59K/K99E, E32Q/D34N/E59K/K99E,E32K/D34N/E59K/K99E, D34N/E59Q, E59Q/E187Q, S43E/E59Q, S43K/E49Q,E59Q/K163E, E59Q/K99E, E59Q/K258E, E59Q/K260E, E59K/K99E,D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y, E59Y/K99Y, E59Y/K99E,E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K, E59K/K99E/R159E,E59K/K99E/K264E, D18K/E59K/K99E/K264E, DI8K/E59K/K99E/C252S,D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S, E59K/K99E/C252S/K264E,E59K/K99E/C252S, N103D/N113D, N103D/N200D, N103D/N281D, N113D/N200D,N113D/N281D, N200D/N281D, N103D/N113D/N200D, N103D/N113D/N281D,N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q, N103Q/N200Q,N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q, N103Q/N113Q/N200Q,N103Q/N113Q/N281Q, N103Q/N200Q/N281Q, N113Q/N200Q/N281Q,N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E; and an IL-12p35 variantcomprising one or more substitutions selected from the group consistingof: N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D,N85Q, L89A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E,N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D,N195Q, N71D/N85D/N195D, N151D/E153Q, N151D/D165N, Q130E/N151D,N151D/K158E, E79Q/N151D, D55Q/N151D, N136D/N151D, N21D/N151D,E143Q/N151D, N71Q/N85Q, N71Q/N195Q, N85Q/N195Q, N71Q/N85Q/N195Q,N71D/N85D, N71D/N195D, and N85D/N195D.

In this format, useful embodiments include, but are not limited to,those found in FIG. 10 .

C. scIL-12-Fc Format

In a further embodiment, the present invention provides the N-terminalsingle-chain IL-12-Fc fusion or “scIL-12-Fc” format. In this embodiment,as shown in FIGS. 8C-D, the heterodimeric fusion protein comprises twomonomers. The first monomer comprises (from N-to-C-terminus)scIL-12-optional domain linker-Fc. The second monomer comprises an“empty” Fc, comprising all or part of the hinge-CH2-CH3. The scIL-12 maybe “scIL-12(p40/p35)”, wherein the IL-12p40 subunit is N-terminallylinked (with or without a domain linker) to the IL-12p35 subunit, or“scIL-12(p35/p40)”, wherein the IL-12p35 is N-terminally linked (with orwithout a domain linker) to the IL-12p40 subunit.

In the scIL-12-Fc format, a preferred embodiment utilizes the skewvariant pair S364K/E357Q:L368D/K370S.

In the scIL-12-Fc format, preferred embodiments are shown in FIG. 11 asXENP24203 (including chain 1 and chain 2) and XENP24204 (including chain1 and chain 2).

In the scIL-12-Fc format, a preferred embodiment is shown in FIG. 50 asXENP31290 (including chain 1 and chain 2).

In some embodiments, the scIL-12-Fc format is a bivalent IL-12-Fcfusion. In some embodiments, the bivalent IL-12-Fc fusion format (FIGS.48A-B) comprises two identical monomers each comprising a scIL-12complex recombinant fused to the N-terminus of a homodimeric Fc chain(optionally via a domain linker).

In some embodiments, the scIL-12-Fc format a heterodimeric Fc fusionprotein comprising: a) a first monomer comprising, from N- toC-terminal: i) a IL-12p40 domain protein; ii) an optional first domainlinker; iii) a a IL-12p35 domain protein; iv) an optional second linker;v) a first variant Fc domain protein; and b) a second monomer comprisinga second variant Fc domain protein.

In some embodiments, the scIL-12-Fc format a heterodimeric Fc fusionprotein comprising: a) a first monomer comprising, from N- toC-terminal: i) a IL-12p35 domain protein; ii) an optional first domainlinker; iii) a a IL-12p40 domain protein; iv) an optional second linker;v) a first variant Fc domain protein; and b) a second monomer comprisinga second variant Fc domain protein.

In some embodiments, the Fc variants comprise one or more skew, pI, andablation variants as provided herein. In one embodiment, Fc variantscomprise particular skew, pI, and ablation variants. In someembodiments, modifications promoting heterodimerization of the first andthe second Fc domains are a set of amino acid substitutions selectedfrom the group consisting of L368D/K370S and S364K; L368D/K370S andS364K/E357L; L368D/K370S and S364K/E357Q; T411E/K360E/Q362E and D401K;L368E/K370S and S364K; K370S and S364K/E357Q; T366S/L368A/Y407V andT366W; T366S/L368A/Y407V/Y349C and T366W/S354C, according to EUnumbering. In some embodiments, the first and/or the second Fc domainshave an additional set of amino acid substitutions comprisingQ295E/N384D/Q418E/N421D, according to EU numbering. In some embodiments,the first and/or the second Fc domains have an additional set of aminoacid substitutions selected from the group consisting of G236R/L328R,E233P/L234V/L235A/G236_/S239K, E233P/L234V/L235A/G236_/S239K/A327G,E233P/L234V/L235A/G236_/S267K/A327G, E233P/L234V/L235A/G236_, andE233P/L234V/L235A/G236_/S267K, according to EU numbering. In someembodiments the first and second Fc domains further comprise amino acidsubstitutions M428L/N434S.

In this format, useful IL-12p40 protein domains include, but are notlimited to, SEQ ID NO: 4 (human IL-12 subunit beta (IL-12p40) matureform sequence). In this format, useful IL-12p40 variants include, butare not limited to, E59K, E59Q, D18N, D18K, E32Q, E33Q, D34N, D34K,Q42E, S43E, S43K, E45Q, Q56E, D62N, E73Q, D87N, K99E, K99Y, E100Q,N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D,N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E,N281D, N281Q, and E299Q. In this format, useful IL-12p40 variantsinclude, but are not limited to, N103D/N113D/N200D/N281D, Q42E/E45Q,E45Q/Q56E, Q42E/E59Q, Q56E/E59Q, Q42E/E45Q/Q56E, E45Q/Q56E/E59Q,E32Q/E59Q, D34N/E59K, D34N/E59K/K99E, D34K/E59K/K99E,E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q, E59Q/E187Q,S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E, E59Q/K260E,E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y, E59Y/K99Y,E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In this format, IL-12p35 protein domains include SEQ ID NO: 2 (humanIL-12 subunit alpha (IL-12p35) mature form sequence). In this format,useful IL-12p35 variants, include, but are not limited to, N21D, Q35D,E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A,M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K,E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q. Inthis format, useful IL-12p35 variants, include, but are not limited to,N71D/N85D/N195D, N151D/E153Q, N151D/D165N, Q130E/N151D, N151D/K158E,E79Q/N151D, D55Q/N151D, N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q,N71Q/N195Q, N85Q/N195Q, N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, andN85D/N195D.

In this format, useful combinations of a variant IL-12p40 subunit andIL-12p35 subunit include, but are not limited to IL-12p40(E59K/K99Y) andIL-12p35(SEQ ID NO: 2), IL-12p40(D18K/E59K/K99E) and IL-12p35 (SEQ IDNO: 2), IL-12p40(E59K/K99E/K264E) and IL-12p35(SEQ ID NO: 2),IL-12p40(D18K/E59K/K99E/K264E) and IL-12p35(SEQ ID NO: 2),IL-12p40(D18K/E59K/K99E/C252S) and IL-12p35(SEQ ID NO: 2),IL-12p40(D18K/E59K/K99E/C252S/K264E) and IL-12p35(SEQ ID NO: 2),IL-12p40(E59K/K99Y/C252S) and IL-12p35(SEQ ID NO: 2),IL-12p40(E59K/K99E/C252S/K264E) and IL-12p35(SEQ ID NO: 2), andIL-12p40(E59K/K99E/C252S) and IL-12p35(SEQ ID NO: 2).

In this format, useful combinations of a variant IL-12p40 subunit and aIL-12p35 subunit include, but are not limited to a combination of anIL-12p35 comprising SEQ ID NO: 2 (human IL-12 subunit alpha (IL-12p35)mature form sequence) and a variantIL-12p40 comprising one or moresubstitutions including, but not limited, to E59K, E59Q, D18N, D18K,E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, D62N, E73Q, D87N,K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E,K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E,K260E, E262Q, K264E, N281D, N281Q, E299Q, N103D/N113D/N200D/N281D,Q42E/E45Q, E45Q/Q56E, Q42E/E59Q, Q56E/E59Q, Q42E/E45Q/Q56E,E45Q/Q56E/E59Q, E32Q/E59Q, D34N/E59K, D34N/E59K/K99E, D34K/E59K/K99E,E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q, E59Q/E187Q,S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E, E59Q/K260E,E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y, E59Y/K99Y,E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In this format, useful combinations of IL-12p40 and IL-12p35 variantsinclude, but are not limited to a combination of an IL-12p40 comprisingSEQ ID NO: 4 (human IL-12 subunit beta (IL-12p40) mature form sequence)and an IL-12p35 comprising N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q,L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A, L124A, M125A, Q130E,Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q,E163Q, D165N, I171A, N195D, N195Q, N71D/N85D/N195D, N151D/E153Q,N151D/D165N, Q130E/N151D, N151D/K158E, E79Q/N151D, D55Q/N151D,N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q, N71Q/N195Q, N85Q/N195Q,N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, and N85D/N195D.

In this format, useful combinations of IL-12p40 variants and IL-12p35variants include, but are not limited to, a combination of an IL-12p40variant comprising one or more amino acid substitution selected from thegroup consisting of: E59K, E59Q, D18N, D18K, E32Q, E33Q, D34N, D34K,Q42E, S43E, S43K, E45Q, Q56E, D62N, E73Q, D87N, K99E, K99Y, E100Q,N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D,N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E,N281D, N281Q, E299Q, N103D/N113D/N200D/N281D, Q42E/E45Q, E45Q/Q56E,Q42E/E59Q, Q56E/E59Q, Q42E/E45Q/Q56E, E45Q/Q56E/E59Q, E32Q/E59Q,D34N/E59K, D34N/E59K/K99E, D34K/E59K/K99E, E32Q/D34N/E59K/K99E,E32K/D34N/E59K/K99E, D34N/E59Q, E59Q/E187Q, S43E/E59Q, S43K/E49Q,E59Q/K163E, E59Q/K99E, E59Q/K258E, E59Q/K260E, E59K/K99E,D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y, E59Y/K99Y, E59Y/K99E,E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K, E59K/K99E/R159E,E59K/K99E/K264E, D18K/E59K/K99E/K264E, DI8K/E59K/K99E/C252S,D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S, E59K/K99E/C252S/K264E,E59K/K99E/C252S, N103D/N113D, N103D/N200D, N103D/N281D, N113D/N200D,N113D/N281D, N200D/N281D, N103D/N113D/N200D, N103D/N113D/N281D,N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q, N103Q/N200Q,N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q, N103Q/N113Q/N200Q,N103Q/N113Q/N281Q, N103Q/N200Q/N281Q, N113Q/N200Q/N281Q,N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E; and an IL-12p35 variantcomprising one or more substitutions selected from the group consistingof: N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D,N85Q, L89A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E,N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D,N195Q, N71D/N85D/N195D, N151D/E153Q, N151D/D165N, Q130E/N151D,N151D/K158E, E79Q/N151D, D55Q/N151D, N136D/N151D, N21D/N151D,E143Q/N151D, N71Q/N85Q, N71Q/N195Q, N85Q/N195Q, N71Q/N85Q/N195Q,N71D/N85D, N71D/N195D, and N85D/N195D.

In this format, useful embodiments include, but are not limited to,those found in FIG. 11 .

D. Fc-scIL-12 Format

In an additional embodiment, the present invention provides theC-terminal single-chain IL-12-Fc fusion or “Fc-scIL-12” format. In thisembodiment, as shown in FIGS. 8E-F, the heterodimeric fusion proteincomprises two monomers. The first monomer comprises (fromN-to-C-terminus) Fc-optional linker-scIL-12. The second monomercomprises an “empty” Fc, comprising all or part of the hinge-CH2-CH3. Asabove, the scIL-12 may be “scIL-12(p40/p35)”, wherein the IL-12p40subunit is N-terminally linked (with or without a domain linker) to theIL-12p35 subunit, or “scIL-12(p35/p40)”, wherein the IL-12p35 isN-terminally linked (with or without a domain linker) to the IL-12p40subunit.

In the Fc-scIL-12 format, a preferred embodiment utilizes the skewvariant pair S364K/E357Q:L368D/K370S.

In some embodiments, the scIL-12-Fc format is a bivalent IL-12-Fcfusion. In some embodiments, the bivalent IL-12-Fc fusion format (FIGS.48C-D) comprises two identical monomers each comprising a scIL-12complex recombinant fused to the C-terminus of a homodimeric Fc chain(optionally via a domain linker).

In some embodiments, the scIL-12-Fc format provides a heterodimeric Fcfusion protein comprising: a) a first monomer comprising, from N- toC-terminal: i) a first variant Fc domain protein; ii) an optional firstdomain linker; iii) a IL-12p40 domain protein; iv) an optional seconddomain linker; v) a IL-12p35 domain protein; and b) a second monomercomprising a second variant Fc domain protein.

In some embodiments, the scIL-12-Fc format provides a heterodimeric Fcfusion protein comprising: a) a first monomer comprising, from N- toC-terminal: i) a first variant Fc domain protein; ii) an optional firstdomain linker; iii) a IL-12p35 domain protein; iv) an optional seconddomain linker; v) a IL-12p40 domain protein; and b) a second monomercomprising a second variant Fc domain protein.

In some embodiments, the Fc variants comprise one or more skew, pI, andablation variants as provided herein. In one embodiment, Fc variantscomprise particular skew, pI, and ablation variants. In someembodiments, modifications promoting heterodimerization of the first andthe second Fc domains are a set of amino acid substitutions selectedfrom the group consisting of L368D/K370S and S364K; L368D/K370S andS364K/E357L; L368D/K370S and S364K/E357Q; T411E/K360E/Q362E and D401K;L368E/K370S and S364K; K370S and S364K/E357Q; T366S/L368A/Y407V andT366W; T366S/L368A/Y407V/Y349C and T366W/S354C, according to EUnumbering. In some embodiments, the first and/or the second Fc domainshave an additional set of amino acid substitutions comprisingQ295E/N384D/Q418E/N421D, according to EU numbering. In some embodiments,the first and/or the second Fc domains have an additional set of aminoacid substitutions selected from the group consisting of G236R/L328R,E233P/L234V/L235A/G236_/S239K, E233P/L234V/L235A/G236_/S239K/A327G,E233P/L234V/L235A/G236_/S267K/A327G, E233P/L234V/L235A/G236_, andE233P/L234V/L235A/G236_/S267K, according to EU numbering. In someembodiments the first and second Fc domains further comprise amino acidsubstitutions M428L/N434S.

In this format, useful IL-12p40 protein domains include, but are notlimited to, SEQ ID NO: 4 (human IL-12 subunit beta (IL-12p40) matureform sequence). In this format, useful IL-12p40 variants include, butare not limited to, E59K, E59Q, D18N, D18K, E32Q, E33Q, D34N, D34K,Q42E, S43E, S43K, E45Q, Q56E, D62N, E73Q, D87N, K99E, K99Y, E100Q,N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D,N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E,N281D, N281Q, and E299Q. In this format, useful IL-12p40 variantsinclude, but are not limited to, N103D/N113D/N200D/N281D, Q42E/E45Q,E45Q/Q56E, Q42E/E59Q, Q56E/E59Q, Q42E/E45Q/Q56E, E45Q/Q56E/E59Q,E32Q/E59Q, D34N/E59K, D34N/E59K/K99E, D34K/E59K/K99E,E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q, E59Q/E187Q,S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E, E59Q/K260E,E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y, E59Y/K99Y,E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In this format, IL-12p35 protein domains include SEQ ID NO: 2 (humanIL-12 subunit alpha (IL-12p35) mature form sequence). In this format,useful IL-12p35 variants, include, but are not limited to, N21D, Q35D,E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D, N85Q, L89A, F96A,M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K,E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D, and N195Q. Inthis format, useful IL-12p35 variants, include, but are not limited to,N71D/N85D/N195D, N151D/E153Q, N151D/D165N, Q130E/N151D, N151D/K158E,E79Q/N151D, D55Q/N151D, N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q,N71Q/N195Q, N85Q/N195Q, N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, andN85D/N195D.

In this format, useful combinations of a variant IL-12p40 subunit andIL-12p35 subunit include, but are not limited to IL-12p40(E59K/K99Y) andIL-12p35(SEQ ID NO: 2), IL-12p40(D18K/E59K/K99E) and IL-12p35 (SEQ IDNO: 2), IL-12p40(E59K/K99E/K264E) and IL-12p35(SEQ ID NO: 2),IL-12p40(D18K/E59K/K99E/K264E) and IL-12p35(SEQ ID NO: 2),IL-12p40(D18K/E59K/K99E/C252S) and IL-12p35(SEQ ID NO: 2),IL-12p40(D18K/E59K/K99E/C252S/K264E) and IL-12p35(SEQ ID NO: 2),IL-12p40(E59K/K99Y/C252S) and IL-12p35(SEQ ID NO: 2),IL-12p40(E59K/K99E/C252S/K264E) and IL-12p35(SEQ ID NO: 2), andIL-12p40(E59K/K99E/C252S) and IL-12p35(SEQ ID NO: 2).

In this format, useful combinations of a variant IL-12p40 subunit and aIL-12p35 subunit include, but are not limited to a combination of anIL-12p35 comprising SEQ ID NO: 2 (human IL-12 subunit alpha (IL-12p35)mature form sequence) and a variantIL-12p40 comprising one or moresubstitutions including, but not limited, to E59K, E59Q, D18N, D18K,E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, D62N, E73Q, D87N,K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E,K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E,K260E, E262Q, K264E, N281D, N281Q, E299Q, N103D/N113D/N200D/N281D,Q42E/E45Q, E45Q/Q56E, Q42E/E59Q, Q56E/E59Q, Q42E/E45Q/Q56E,E45Q/Q56E/E59Q, E32Q/E59Q, D34N/E59K, D34N/E59K/K99E, D34K/E59K/K99E,E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q, E59Q/E187Q,S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E, E59Q/K260E,E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y, E59Y/K99Y,E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In this format, useful combinations of IL-12p40 and IL-12p35 variantsinclude, but are not limited to a combination of an IL-12p40 comprisingSEQ ID NO: 4 (human IL-12 subunit beta (IL-12p40) mature form sequence)and an IL-12p35 comprising N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q,L75A, N76D, E79Q, N85D, N85Q, L89A, F96A, M97A, L124A, M125A, Q130E,Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q,E163Q, D165N, I171A, N195D, N195Q, N71D/N85D/N195D, N151D/E153Q,N151D/D165N, Q130E/N151D, N151D/K158E, E79Q/N151D, D55Q/N151D,N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q, N71Q/N195Q, N85Q/N195Q,N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, and N85D/N195D.

In this format, useful combinations of IL-12p40 variants and IL-12p35variants include, but are not limited to, a combination of an IL-12p40variant comprising one or more amino acid substitution selected from thegroup consisting of: E59K, E59Q, D18N, D18K, E32Q, E33Q, D34N, D34K,Q42E, S43E, S43K, E45Q, Q56E, D62N, E73Q, D87N, K99E, K99Y, E100Q,N103D, N103Q, N113D, N113Q, Q144E, D161N, R159E, K163E, E187Q, N200D,N200Q, N218Q, Q229E, E235Q, C252S, Q256N, K258E, K260E, E262Q, K264E,N281D, N281Q, E299Q, N103D/N113D/N200D/N281D, Q42E/E45Q, E45Q/Q56E,Q42E/E59Q, Q56E/E59Q, Q42E/E45Q/Q56E, E45Q/Q56E/E59Q, E32Q/E59Q,D34N/E59K, D34N/E59K/K99E, D34K/E59K/K99E, E32Q/D34N/E59K/K99E,E32K/D34N/E59K/K99E, D34N/E59Q, E59Q/E187Q, S43E/E59Q, S43K/E49Q,E59Q/K163E, E59Q/K99E, E59Q/K258E, E59Q/K260E, E59K/K99E,D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y, E59Y/K99Y, E59Y/K99E,E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K, E59K/K99E/R159E,E59K/K99E/K264E, D18K/E59K/K99E/K264E, DI8K/E59K/K99E/C252S,D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S, E59K/K99E/C252S/K264E,E59K/K99E/C252S, N103D/N113D, N103D/N200D, N103D/N281D, N113D/N200D,N113D/N281D, N200D/N281D, N103D/N113D/N200D, N103D/N113D/N281D,N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q, N103Q/N200Q,N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q, N103Q/N113Q/N200Q,N103Q/N113Q/N281Q, N103Q/N200Q/N281Q, N113Q/N200Q/N281Q,N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E; and an IL-12p35 variantcomprising one or more substitutions selected from the group consistingof: N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D, E79Q, N85D,N85Q, L89A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D, E143Q, Q146E,N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N, I171A, N195D,N195Q, N71D/N85D/N195D, N151D/E153Q, N151D/D165N, Q130E/N151D,N151D/K158E, E79Q/N151D, D55Q/N151D, N136D/N151D, N21D/N151D,E143Q/N151D, N71Q/N85Q, N71Q/N195Q, N85Q/N195Q, N71Q/N85Q/N195Q,N71D/N85D, N71D/N195D, and N85D/N195D.

VIII. Useful Embodiments of the Invention

As will be appreciated by those in the art and discussed more fullybelow, the heterodimeric fusion proteins of the present invention cantake on a wide variety of configurations, as are generally depicted inFIGS. 8A-F. The amino acid sequences of exemplary fusion proteins areprovided in FIGS. 9-11 .

Many of the embodiments outlined herein rely in general on the formatcomprising a first monomer (first fusion protein) comprising an IL-12p35subunit domain covalently attached (optionally via a domain linker) tothe N-terminus of a first Fc domain, and a second monomer (second fusionprotein) comprising an IL-12p40 subunit domain covalently attached(optionally via a domain linker) to the N-terminus of a second Fcdomain. Exemplary embodiments of this format include, but are notlimited to XENP27201. In additional embodiments, the first monomer has apolypeptide sequence selected from the group consisting of i) SEQ IDNO:47 (XENP27201 Chain 1), ii) SEQ ID NO:85 (XenD24752), iii) SEQ IDNO:86 (XenD24753), iv) SEQ ID NO:87 (XenD24754), v) SEQ ID NO:88(XenD24755), vi) SEQ ID NO:89 (XenD24756), vii) SEQ ID NO:90(XenD24757), viii) SEQ ID NO:91 (XenD24758), ix) SEQ ID NO:92(XenD24759), x) SEQ ID NO:93 (XenD24760), xi) SEQ ID NO:94 (XenD24761),xii) SEQ ID NO:95 (XenD24762), xiii) SEQ ID NO:96 (XenD24763), xiv) SEQID NO:97 (XenD24764), xv) SEQ ID NO:98 (XenD24765), xvi) SEQ ID NO:99(XenD24766), xvii) SEQ ID NO:100 (XenD24767), xviii) SEQ ID NO:101(XenD24768), xix) SEQ ID NO:102 (XenD24769), xx) SEQ ID NO:103(XenD24770), xxi) SEQ ID NO:104 (XenD24771), xxii) SEQ ID NO:105(XenD24772), xxiii) SEQ ID NO:106 (XenD24773), xxiv) SEQ ID NO:107(XenD24774), xxv) SEQ ID NO:108 (XenD24775), xxvi) SEQ ID NO:109(XenD24776), xxvii) SEQ ID NO:110 (XenD24777), xxviii) SEQ ID NO:111(XenD24778), xxix) SEQ ID NO:112 (XenD24792), xxx) SEQ ID NO:215(XenD25922), xxxi) SEQ ID NO:216 (XenD25923), xxxii) SEQ ID NO:217(XenD25924), xxxiii) SEQ ID NO:218 (XenD25925), xxxiv) SEQ ID NO:219(XenD25926), xxxv) SEQ ID NO:220 (XenD25927), xxxvi) SEQ ID NO:221(XenD25928), xxxvii) SEQ ID NO:222 (XenD25929), xxxviii) SEQ ID NO:223(XenD25930), and xxxix) SEQ ID NO:224 (XenD25931); and the secondmonomer has a polypeptide sequence selected from the group consisting ofi) SEQ ID NO:48 (XENP27201 Chain 2), ii) SEQ ID NO:126 (XenD24779), iii)SEQ ID NO:127 (XenD24780), iv) SEQ ID NO:128 (XenD24781), v) SEQ IDNO:129 (XenD24782), vi) SEQ ID NO:130 (XenD24783), vii) SEQ ID NO:131(XenD24784), viii) SEQ ID NO:132 (XenD24785), ix) SEQ ID NO:133(XenD24786), x) SEQ ID NO:134 (XenD24787), xi) SEQ ID NO:135(XenD24788), xii) SEQ ID NO:136 (XenD24789), xiii) SEQ ID NO:137(XenD24790), xiv) SEQ ID NO:138 (XenD24791), xv) SEQ ID NO:236(XenD25911), xvi) SEQ ID NO:237 (XenD25912), xvii) SEQ ID NO:238(XenD25913), xviii) SEQ ID NO:239 (XenD25914), xix) SEQ ID NO:240(XenD25915), xx) SEQ ID NO:241 (XenD25916), xxi) SEQ ID NO:242(XenD25917), xxii) SEQ ID NO:243 (XenD25918), xxiii) SEQ ID NO:244(XenD25919), xxiv) SEQ ID NO:245 (XenD25920), and xxv) SEQ ID NO:246(XenD25921). Particular such embodiments include, but are not limitedto, XENP27201, XENP28820, XENP28821, XENP28822, XENP28823, XENP28824,XENP28825, XENP28826, XENP28827, XENP28828, XENP28829, XENP28830,XENP28831, XENP28832, XENP28833, XENP28834, XENP28835, XENP28836,XENP28837, XENP28838, XENP28839, XENP28840, XENP28841, XENP28842,XENP28843, XENP28844, XENP28845, XENP28846, XENP28847, XENP28848,XENP28849, XENP28850, XENP28851, XENP28852, XENP29949, XENP29950,XENP29951, XENP29952, XENP30597, XENP30598, XENP30599, XENP30600,XENP30601, XENP30602, XENP30603, XENP30604, XENP30605, XENP30606.XENP30307, XENP30308, XENP30609, XENP31250, XENP31251, XENP31252,XENP31253, XENP31254, XENP31255, XENP31256, XENP31257, XENP31258,XENP31259, XENP31260, XENP31261, XENP31262, XENP31263, XENP31264,XENP31265, XENP31286, XENP31142, XENP31143, XENP31144, XENP31145,XENP31146, XENP31582, XENP31583, XENP31584, XENP32187, XENP32188,XENP32189, XENP32190, XENP32191, XENP32991, XENP32992, XENP32993,XENP32994, XENP32995, XENP32996, XENP32997, XENP32998, XENP32999,XENP33000, XENP33001, XENP33002, XENP33003, XENP33004, XENP33005,XENP33006, XENP33007, XENP33008, XENP33008, XENP33009, XENP33010, andXENP33011. A useful embodiment of a heterodimer Fc fusion proteincomprises a first monomer (first fusion protein) comprising an IL-12p35subunit domain covalently attached (optionally via a domain linker) tothe C-terminus of a first Fc domain, and a second monomer (second fusionprotein) comprising an IL-12p40 subunit domain covalently attached(optionally via a domain linker) to the C-terminus of a second Fcdomain. Exemplary embodiments of this format include, but are notlimited to XENP27202.

Another useful embodiment of a heterodimer Fc fusion protein comprises afirst monomer (first fusion protein) comprising a single-chain IL-12complex (“scIL-12”) covalently attached (optionally via a domain linker)to the N-terminus of a first Fc domain, and a second Fc domain (e.g., anempty Fc domain). The scIL-12 may be “scIL-12(p40/p35)”, wherein theIL-12p40 subunit is N-terminally linked (with or without a domainlinker) to the IL-12p35 subunit, or “scIL-12(p35/p40)”, wherein theIL-12p35 is N-terminally linked (with or without a domain linker) to theIL-12p40 subunit. Exemplary embodiments of this format includes, but isnot limited to, XENP27203 and XENP27204.

A further useful embodiment of a heterodimer Fc fusion protein comprisesa first monomer (first fusion protein) comprising a single-chain IL-12complex (“scIL-12”) covalently attached (optionally via a domain linker)to the C-terminus of a first Fc domain, and a second Fc domain (e.g., anempty Fc domain). The scIL-12 may be “scIL-12(p40/p35)”, wherein theIL-12p40 subunit is N-terminally linked (with or without a domainlinker) to the IL-12p35 subunit, or “scIL-12(p35/p40)”, wherein theIL-12p35 is N-terminally linked (with or without a domain linker) to theIL-12p40 subunit.

For any of the heterodimer Fc fusion proteins outlined herein, theoptional domain linkers used on the first monomer, on the secondmonomer, and/or in the scIL-12 can be the same or different. Inaddition, the first Fc domain and the second Fc domain of theheterodimeric protein can have different amino acid sequences.

The Fc domains of the present invention comprise IgG Fc domains, e.g.,IgG1 Fc domains. In some embodiments, the first and second Fc domainscomprising a set of amino acid substitutions selected from the groupconsisting of: L368D/K370S and S364K; L368D/K370S and S364K/E357L;L368D/K370S and S364K/E357Q; T411E/K360E/Q362E and D401K; L368E/K370Sand S364K; K370S and S364K/E357Q and T366S/L368A/Y407V:T366W (optionallyincluding a bridging disulfide, T366S/L368A/Y407V/Y349C:T366W/S354C),according to EU numbering. In some instances, the first and/or thesecond Fc domains of any of the heterodimeric Fc fusion formats outlinedherein can have an additional set of amino acid substitutions comprisingQ295E/N384D/Q418E/N421D, according to EU numbering. In some embodiments,the first and/or the second Fc domains have an additional set of aminoacid substitutions selected from the group consisting of G236R/L328R,E233P/L234V/L235A/G236_/S239K, E233P/L234V/L235A/G236_/S239K/A327G,E233P/L234V/L235A/G236_/S267K/A327G, E233P/L234V/L235A/G236_, andE233P/L234V/L235A/G236_/S267K, according to EU numbering.

Additional heterodimerization or homodimerization variants can beindependently and optionally included and selected from variantsoutlined in the figures. These compositions can further compriseablation variants, pI variants, charged variants, isotypic variants,etc.

A. IL-12p40 Variants

In some embodiments, the IL-12p40 subunit is a variant IL-12p40 subunit.In some particular such embodiments, the IL-12p40 subunit is a variantIL-12p40 subunit having reduced heterogeneity. In other particular suchembodiments, the IL-12p40 subunit is a variant IL-12p40 subunit havingaltered, that is either reduced or increased, affinity for IL-12receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2(IL-12Rβ2), and/or IL-12 receptor complex. In some embodiments, thevariant IL-12p40 subunit has one or more amino acid modifications atamino acid residues selected from the group consisting of E3, D7, E12,D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32, E33, D34, L40,D41, Q42, S43, E45, L47, T54, 155, Q56, K58, E59, F60, G61, D62, Q65,Y66, E73, K84, E86, D87, G88, 189, W90, D93, D97, K99, E100, K102, N103,K104, F106, E110, N113, Y114, D129, D142, Q144, E156, R159, D161, N162,K163, D166, D170, Q172, D174, A176, C177, P178, A179, A180, E181, S183,P185, E187, N200, S204, F206, R208, D209, D214, N218, Q220, N226, Q229,E231, E235, T242, P243, S245, Y246, F247, S248, C252, Q256, K258, K260,E262, K264, D265, D270, N281, Q289, D290, R291, Y292, Y293, and E299.(numbered according to the human IL-12 subunit beta (IL-12p40) matureform sequence).

In some embodiments, the variant IL-12p40 subunit has one or more aminoacid substitutions selected from the group consisting of D18N, D18K,E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45Q, Q56E, E59Q, E59K, D62N,E73Q, D87N, K99E, K99Y, E100Q, N103D, N103Q, N113D, N113Q, Q144E, D161N,R159E, K163E, E187Q, N200D, N200Q, N218Q, Q229E, E235Q, C252S, Q256N,K258E, K260E, E262Q, K264E, N281D, N281Q, and E299Q. In someembodiments, the variant IL-12p40 subunit has amino acid substitutionsselected from the group consisting of N103D/N113D/N200D/N281D,Q42E/E45Q, E45Q/Q56E, Q42E/E59Q, Q56E/E59Q, Q42E/E45Q/Q56E,E45Q/Q56E/E59Q, E32Q/E59Q, D34N/E59K, D34N/E59K/K99E, D34K/E59K/K99E,E32Q/D34N/E59K/K99E, E32K/D34N/E59K/K99E, D34N/E59Q, E59Q/E187Q,S43E/E59Q, S43K/E49Q, E59Q/K163E, E59Q/K99E, E59Q/K258E, E59Q/K260E,E59K/K99E, D18K/E59K/K99E, E59K/K99E/K264E, E59K/K99Y, E59Y/K99Y,E59Y/K99E, E45K/E59K/K99E, E59K/K99E/Q144E, E59K/K99E/Q144K,E59K/K99E/R159E, E59K/K99E/K264E, D18K/E59K/K99E/K264E,DI8K/E59K/K99E/C252S, D18K/E59K/K99E/C252S/K264E, E59K/K99Y/C252S,E59K/K99E/C252S/K264E, E59K/K99E/C252S, N103D/N113D, N103D/N200D,N103D/N281D, N113D/N200D, N113D/N281D, N200D/N281D, N103D/N113D/N200D,N103D/N113D/N281D, N103D/N200D/N281D, N113D/N200D/N281D, N103Q/N113Q,N103Q/N200Q, N103Q/N281Q, N113Q/N200Q, N113Q/N281Q, N200Q/N281Q,N103Q/N113Q/N200Q, N103Q/N113Q/N281Q, N103Q/N200Q/N281Q,N113Q/N200Q/N281Q, N103Q/N113Q/N200Q/N281Q, E59K/K99E/N103Q/C252S/K264E,E59K/K99E/N113Q/C252S/K264E, E59K/K99E/N200Q/C252S/K264E,E59K/K99E/N281Q/C252S/K264E, E59K/K99E/N103Q/N113Q/C252S/K264E,E59K/K99E/N103Q/N200Q/C252S/K264E, E59K/K99E/N103Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/C252S/K264E, E59K/K99E/N113Q/N281Q/C252S/K264E,E59K/K99E/N200Q/N281Q/C252S/K264E,E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E,E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E,E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E, andE59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E.

In some embodiments, the IL-12p40 variant has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:57(IL-12p40(N103D)), ii) SEQ ID NO:58 (IL-12p40(N113D)), iii) SEQ ID NO:59(IL-12p40(N200D)), iv) SEQ ID NO:60 (IL-12p40(N281D)), v) SEQ ID NO:61(IL-12p40(N103D/N113D/N200D/N281D)), vi) SEQ ID NO:62 (IL-12p40(Q42E)),vii) SEQ ID NO:63 (IL-12p40(E45Q)), viii) SEQ ID NO:64 (IL-12p40(Q56E)),ix) SEQ ID NO:65 (IL-12p40(E59Q)), x) SEQ ID NO:66 (IL-12p40(D62N)), xi)SEQ ID NO:67 (IL-12p40(Q42E/E45Q)), xii) SEQ ID NO:68(IL-12p40(E45Q/Q56E)), xiii) SEQ ID NO:69 (IL-12p40(Q42E/E59Q)), xiv)SEQ ID NO:70 (IL-12p40(Q56E/E59Q)), xv) SEQ ID NO:71(IL-12p40(Q42E/E45Q/Q56E)), xvi) SEQ ID NO:72(IL-12p40(E45Q/Q56E/E59Q)), xvii) SEQ ID NO:73 (IL-12p40(D161N)), xviii)SEQ ID NO:74 (IL-12p40(E73Q)), xix) SEQ ID NO:75 (IL-12p40(Q144E)), xx)SEQ ID NO:76 (IL-12p40(E262Q)), xxi) SEQ ID NO:77 (IL-12p40(E100Q)),xxii) SEQ ID NO:78 (IL-12p40(D18N)), xxiii) SEQ ID NO:79(IL-12p40(E33Q)), xxiv) SEQ ID NO:80 (IL-12p40(Q229E)), xxv) SEQ IDNO:81 (IL-12p40(E235Q)), xxvi) SEQ ID NO:82 (IL-12p40(Q256N)), xxvii)SEQ ID NO:83 (IL-12p40(E299Q)), xxviii) SEQ ID NO:84 (IL-12p40(D87N)),xxix) IL-12p40(E32Q), xxx) IL-12p40(D34N), xxxi) IL-12p40(S43E), xxxii)IL-12p40(S43K), xxxiii) SEQ ID NO:379 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/N281Q/C252S/K264E)), xxxiv) SEQ ID NO:205(IL-12p40(E59K)), xxxv) IL-12p40(K99E), xxxvi) IL-12p40(K163E), xxxvii)IL-12p40(E187Q), xxxviii) IL-12p40(K258E), xxxix) IL-12p40(K260E), xl)SEQ ID NO:206 (IL-12p40(E32Q/E59Q)), xli) SEQ ID NO:207(IL-12p40(D34N/E59Q)), xlii) SEQ ID NO:208 (IL-12p40(E59Q/E187Q)),xliii) SEQ ID NO:209 (IL-12p40(S43E/E59Q)), xliv) SEQ ID NO:210(IL-12p40(S43K/E49Q)), xlv) SEQ ID NO:211 (IL-12p40(E59Q/K163E)), xlvi)SEQ ID NO:212 (IL-12p40(E59Q/K99E)), xlvii) SEQ ID NO:213(IL-12p40(E59Q/K258E)), xlviii) SEQ ID NO:214 (IL-12p40(E59Q/K260E)),xlix) SEQ ID NO: 326 (IL-12p40 (D34N/E59K)), 1) SEQ ID NO: 325(IL-12p40(E59K/K99E)), li) SEQ ID NO: 339 (IL-12p40(D18K/E59K/K99E)),lii) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), liii) SEQ ID NO: 336(IL-12p40 (E59K/K99Y)), liv) SEQ ID NO: 335 (IL-12p40 (E59Y/K99E)), lv)SEQ ID NO: 338 (IL-12p40 (E45K/E59K/K99E)), lvi) SEQ ID NO: 340(IL-12p40 (E59K/K99E/Q144E)), lvii) SEQ ID NO: 341 (IL-12p40(E59K/K99E/Q144K)), lviii) SEQ ID NO: 342 (IL-12p40 (E59K/K99E/R159E)),lix) SEQ ID NO: 343 (IL-12p40 (E59K/K99E/K264E)), lx) SEQ ID NO: 344(IL-12p40 (D18K/E59K/K99E/K264E)), lxi) SEQ ID NO: 360 (IL-12p40(C252S)), lxii) SEQ ID NO: 361 (IL-12p40 (DI8K/E59K/K99E/C252S)), lxiii)SEQ ID NO: 362 (IL-12p40 (D18K/E59K/K99E/C252S/K264E)), lxiv) SEQ ID NO:363 (IL-12p40 (E59K/K99Y/C252S)), lxv) SEQ ID NO: 364 (IL-12p40(E59K/K99E/C252S/K264E)), lxvi) SEQ ID NO: 365 (IL-12p40(E59K/K99E/C252S)), lxvii) SEQ ID NO: 254 (IL-12p40 (N103D/N113D)),lxviii) SEQ ID NO: 255 (IL-12p40 (N103D/N200D)), lxix) SEQ ID NO: 256(IL-12p40 (N103D/N281D)), lxx) SEQ ID NO: 257 (IL-12p40 (N113D/N200D)),lxxi) SEQ ID NO: 258 (IL-12p40 (N113D/N281D)), lxxii) SEQ ID NO: 259(IL-12p40 (N200D/N281D)), lxxiii) SEQ ID NO: 260 (IL-12p40(N103D/N113D/N200D)), lxxiv) SEQ ID NO: 261 (IL-12p40(N103D/N113D/N281D)), lxxv) SEQ ID NO: 262 (IL-12p40(N103D/N200D/N281D)), lxxvi) SEQ ID NO: 263 (IL-12p40(N113D/N200D/N281D)), lxxvii) SEQ ID NO: 264 (IL-12p40 (N103Q)),lxxviii) SEQ ID NO: 265 (IL-12p40 (N113Q)), lxxix) SEQ ID NO: 266(IL-12p40 (N200Q)), lxxx) SEQ ID NO: 267 (IL-12p40 (N281Q)), lxxxi) SEQID NO: 268 (IL-12p40 (N103Q/N113Q)), lxxxii) SEQ ID NO: 269 (IL-12p40(N103Q/N200Q)), lxxxiii) SEQ ID NO: 270 (IL-12p40 (N103Q/N281Q)),lxxxiv) SEQ ID NO: 271 (IL-12p40 (N113Q/N200Q)), lxxxv) SEQ ID NO: 272(IL-12p40 (N113Q/N281Q)), lxxxvi) SEQ ID NO: 273 (IL-12p40(N200Q/N281Q)), lxxxvii) SEQ ID NO: 274 (IL-12p40 (N103Q/N113Q/N200Q)),lxxxviii) SEQ ID NO: 275 (IL-12p40 (N103Q/N113Q/N281Q)), lxxxix) SEQ IDNO: 276 (IL-12p40 (N103Q/N200Q/N281Q)), xc) SEQ ID NO: 277 (IL-12p40(N113Q/N200Q/N281Q)), xci) SEQ ID NO:278 (IL-12p40(N103Q/N113Q/N200Q/N281Q)), xcii) SEQ ID NO:327 (IL-12p40(D34N/E59K/K99E)), xciii) SEQ ID NO:328 (IL-12p40 (D34K/E59K/K99E)),xciv) SEQ ID NO:329 (IL-12p40 (E32Q/D34N/E59K/K99E)), xcv) SEQ ID NO:331(IL-12p40 (E32K/D34N/E59K/K99E)), xcvi) SEQ ID NO: 337 (IL-12p40(E59Y/K99Y)), xcvii) SEQ ID NO:366 (IL-12p40(E59K/K99E/N103Q/C252S/K264E)), xcviii) SEQ ID NO:367 (IL-12p40(E59K/K99E/N113Q/C252S/K264E)), xcix) SEQ ID NO:368 (IL-12p40(E59K/K99E/N200Q/C252S/K264E)), c) SEQ ID NO:369 (IL-12p40(E59K/K99E/N281Q/C252S/K264E)), ci) SEQ ID NO:370 (IL-12p40(E59K/K99E/N103Q/N113Q/C252S/K264E)), cii) SEQ ID NO:371 (IL-12p40(E59K/K99E/N103Q/N200Q/C252S/K264E)), ciii) SEQ ID NO:372 (IL-12p40(E59K/K99E/N103Q/N281Q/C252S/K264E)), civ) SEQ ID NO:373 (IL-12p40(E59K/K99E/N113Q/N200Q/C252S/K264E)), cv) SEQ ID NO:374 (IL-12p40(E59K/K99E/N113Q/N281Q/C252S/K264E)), cvi) SEQ ID NO:375 (IL-12p40(E59K/K99E/N200Q/N281Q/C252S/K264E)), cvii) SEQ ID NO:376 (IL-12p40(E59K/K99E/N103Q/N113Q/N200Q/C252S/K264E)), cviii) SEQ ID NO:377(IL-12p40 (E59K/K99E/N103Q/N200Q/N281Q/C252S/K264E)), and cix) SEQ IDNO:378 (IL-12p40 (E59K/K99E/N113Q/N200Q/N281Q/C252S/K264E)).

B. IL-12p35 Variants

In some embodiments, the IL-12p35 subunit is a variant IL-12p35 subunit.In some particular such embodiments, the IL-12p35 subunit is a variantIL-12p35 subunit having reduced heterogeneity. In other particular suchembodiments, the IL-12p35 subunit is a variant IL-12p35 subunit havingaltered, that is either reduced or increased, affinity for IL-12receptor subunit beta-1 (IL-12Rβ1), IL-12 receptor subunit beta-2(IL-12Rβ2), and/or IL-12 receptor complex. In some embodiments, thevariant IL-12p35 subunit has one or more amino acid modifications atamino acid residues selected from the group consisting of: Q20, N21,Q35, E38, S44, E45, E46, H49, K54, D55, T59, V60, E61, C63, L64, P65,E67, L68, N71, S73, C74, L75, N76, E79, N85, L89, F96, M97, L124, M125,Q130, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, D165, I171,R181, 1182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, andA196. (numbered according to the Human IL-12 subunit alpha (IL-12p35)mature form sequence). In some embodiments, the variant IL-12p35 subunithas one or more amino acid substitutions selected from the groupconsisting of: N21D, Q35D, E38Q, D55Q, D55K, N71D, N71Q, L75A, N76D,E79Q, N85D, N85Q, L89A, F96A, M97A, L124A, M125A, Q130E, Q135E, N136D,E143Q, Q146E, N151D, N151K, E153K, E153Q, K158E, E162Q, E163Q, D165N,I171A, N195D, and N195Q. In some embodiments, the variant IL-12p35subunit has amino acid substitutions N71D/N85D/N195D, N151D/E153Q,N151D/D165N, Q130E/N151D, N151D/K158E, E79Q/N151D, D55Q/N151D,N136D/N151D, N21D/N151D, E143Q/N151D, N71Q/N85Q, N71Q/N195Q, N85Q/N195Q,N71Q/N85Q/N195Q, N71D/N85D, N71D/N195D, and N85D/N195D. In someembodiments, the variant IL-12p35 subunit has a polypeptide sequenceselected from the group consisting of: i) SEQ ID NO:113(IL-12p35(N71D)), ii) SEQ ID NO:114 (IL-12p35(N85D)), iii) SEQ ID NO:115(IL-12p35(N195D)), iv) SEQ ID NO:116 (IL-12p35(N71D/N85D/N195D)), v) SEQID NO:117 (IL-12p35(E153Q)), vi) SEQ ID NO:118 (IL-12p35(E38Q)), vii)SEQ ID NO:119 (IL-12p35(N151D)), viii) SEQ ID NO:120 (IL-12p35(Q135E)),ix) SEQ ID NO:121 (IL-12p35(Q35D)), x) SEQ ID NO:122 (IL-12p35(Q146E)),xi) SEQ ID NO:123 (IL-12p35(N76D)), xii) SEQ ID NO:124(IL-12p35(E162Q)), xiii) SEQ ID NO:125 (IL-12p35(E163Q)), xiv)IL-12p35(N21D), xv) SEQ ID NO:333 IL-12p35(D55Q), xvi) IL-12p35(E79Q),xvii) IL-12p35(Q130E), xviii) IL-12p35(N136D), xix) IL-12p35(E143Q), xx)SEQ ID NO:227 (IL-12p35(N151K)), xxi) SEQ ID NO:226 (IL-12p35(E153K)),xxii) IL-12p35(K158E), xxiii) IL-12p35(D165N), xxiv) SEQ ID NO:225(IL-12p35(N151D/E153Q)), xxv) SEQ ID NO:228 (IL-12p35(N151D/D165N)),xxvi) SEQ ID NO:229 (IL-12p35(Q130E/N151D)), xxvii) SEQ ID NO:230(IL-12p35(N151D/K158E)), xxviii) SEQ ID NO:231 (IL-12p35(E79Q/N151D)),xxix) SEQ ID NO:232 (IL-12p35(D55Q/N151D)), xxx) SEQ ID NO:233(IL-12p35(N136D/N151D)), xxxi) SEQ ID NO:234 (IL-12p35(N21D/N151D)),xxxii) SEQ ID NO:235 (IL-12p35(E143Q/N151D)), xxxiii) SEQ ID NO: 345(IL-12p35(F96A)), xxxiv) SEQ ID NO: 346 (IL-12p35(M97A)), xxxv) SEQ IDNO: 347 (IL-12p35(L89A)), xxxvi) SEQ ID NO: 348 (IL-12p35(L124A)),xxxvii) SEQ ID NO: 349 (IL-12p35(M125A)), xxxviii) SEQ ID NO: 350(IL-12p35(L75A)), xxxiv) SEQ ID NO: 351 (IL-12p35(I171A)), xxxv) SEQ IDNO: 279 (IL-12p35 (N71Q)), xxxvi) SEQ ID NO: 280 (IL-12p35 (N85Q)),xxxvii) SEQ ID NO: 281 (IL-12p35 (N195Q)), xxxviii) SEQ ID NO: 282(IL-12p35 (N71Q/N85Q)), xxxix) SEQ ID NO: 283 (IL-12p35 (N71Q/N195Q)),lx) SEQ ID NO: 284 (IL-12p35 (N85Q/N195Q), lxi) SEQ ID NO: 285 (IL-12p35(N71Q/N85Q/N195Q)), lxii) SEQ ID NO: 286 (IL-12p35 (N71D/N85D)), lxiii)SEQ ID NO: 287 (IL-12p35 (N71D/N195D), lxiv) SEQ ID NO: 288 (IL-12p35(N85D/N195D)), lxv) SEQ ID NO: 333 (IL-12p35 (D55Q)), and lxvi) SEQ IDNO: 334 (IL-12p35 (D55K)).

C. Additional Options

In some embodiments, the scIL-12-Fc format is a bivalent IL-12-Fcfusion. In some embodiments, the bivalent IL-12-Fc fusion format (FIGS.48A-B) comprises two identical monomers each comprising a scIL-12complex recombinant fused to the N-terminus of a homodimeric Fc chain(optionally via a domain linker). In some embodiments, the bivalentIL-12-Fc fusion format (FIGS. 48C-D) comprises two identical monomerseach comprising a scIL-12 complex recombinant fused to the C-terminus ofa homodimeric Fc chain (optionally via a domain linker). Examplesinclude but are not limited to XENP31289 and XENP31291 (FIG. 49 ).

In some embodiments, the IL-12 Fc fusion proteins are further engineeredfor extending half-life by substitutions comprising M428L and N434S. Insome embodiments, the IL-12 Fc fusion proteins are further engineeredfor extending half-life by substitutions comprising M428L/N434S. Any ofthe IL-12 Fc fusions listed in herein may be engineered for extendinghalf-life. Examples include but are not limited to XENP31582, XENP31583,and XENP31584 (FIG. 65 ).

In some embodiments, examples of heterodimeric Fc fusion proteinsinclude XENP27201, XENP28820, XENP28821, XENP28822, XENP28823,XENP28824, XENP28825, XENP28826, XENP28827, XENP28828, XENP28829,XENP28830, XENP28831, XENP28832, XENP28833, XENP28834, XENP28835,XENP28836, XENP28837, XENP28838, XENP28839, XENP28840, XENP28841,XENP28842, XENP28843, XENP28844, XENP28845, XENP28846, XENP28847,XENP28848, XENP28849, XENP28850, XENP28851, XENP28852, XENP29949,XENP29950, XENP29951, XENP29952, XENP30597, XENP30598, XENP30599,XENP30600, XENP30601, XENP30602, XENP30603, XENP30604, XENP30605,XENP30606. XENP30307, XENP30308, XENP30609, XENP31250, XENP31251,XENP31252, XENP31253, XENP31254, XENP31255, XENP31256, XENP31257,XENP31258, XENP31259, XENP31260, XENP31261, XENP31262, XENP31263,XENP31264, XENP31265, XENP31286, XENP31142, XENP31143, XENP31144,XENP31145, XENP31146, XENP31582, XENP31583, XENP31584, XENP32186,XENP32187, XENP32188, XENP32189, XENP32190, XENP32191, XENP32991,XENP32992, XENP32993, XENP32994, XENP32995, XENP32996, XENP32997,XENP32998, XENP32999, XENP33000, XENP33001, XENP33002, XENP33003,XENP33004, XENP33005, XENP33006, XENP33007, XENP33008, XENP33008,XENP33009, XENP33010, and XENP33011.

In some embodiments, examples of the heterodimeric Fc fusion proteincomprise a first fusion protein having a polypeptide sequence selectedfrom the group consisting of: i) SEQ ID NO:47 (XENP27201 Chain 1), ii)SEQ ID NO:85 (XenD24752), iii) SEQ ID NO:86 (XenD24753), iv) SEQ IDNO:87 (XenD24754), v) SEQ ID NO:88 (XenD24755), vi) SEQ ID NO:89(XenD24756), vii) SEQ ID NO:90 (XenD24757), viii) SEQ ID NO:91(XenD24758), ix) SEQ ID NO:92 (XenD24759), x) SEQ ID NO:93 (XenD24760),xi) SEQ ID NO:94 (XenD24761), xii) SEQ ID NO:95 (XenD24762), xiii) SEQID NO:96 (XenD24763), xiv) SEQ ID NO:97 (XenD24764), xv) SEQ ID NO:98(XenD24765), xvi) SEQ ID NO:99 (XenD24766), xvii) SEQ ID NO:100(XenD24767), xviii) SEQ ID NO:101 (XenD24768), xix) SEQ ID NO:102(XenD24769), xx) SEQ ID NO:103 (XenD24770), xxi) SEQ ID NO:104(XenD24771), xxii) SEQ ID NO:105 (XenD24772), xxiii) SEQ ID NO:106(XenD24773), xxiv) SEQ ID NO:107 (XenD24774), xxv) SEQ ID NO:108(XenD24775), xxvi) SEQ ID NO:109 (XenD24776), xxvii) SEQ ID NO:110(XenD24777), xxviii) SEQ ID NO:111 (XenD24778), xxix) SEQ ID NO:112(XenD24792), xxx) SEQ ID NO:215 (XenD25922), xxxi) SEQ ID NO:216(XenD25923), xxxii) SEQ ID NO:217 (XenD25924), xxxiii) SEQ ID NO:218(XenD25925), xxxiv) SEQ ID NO:219 (XenD25926), xxxv) SEQ IDNO:220(XenD25927), xxxvi) SEQ ID NO:221 (XenD25928), xxxvii) SEQ IDNO:222 (XenD25929), xxxviii) SEQ ID NO:223 (XenD25930), xxxix) SEQ IDNO:224 (XenD25931), xl) SEQ ID NO:291 (XenD26411), xli) SEQ ID NO:292(XenD26412), xlii) SEQ ID NO:293 (XenD26413), xliii) SEQ ID NO:294(XenD26414), xliv) SEQ ID NO:295 (XenD26415), xlv) SEQ ID NO:296(XenD26416), xlvi) SEQ ID NO:297 (XenD26417), xlvii) SEQ ID NO:298(XenD26418), xlviii) SEQ ID NO:301 (XenD27070), xlix) SEQ ID NO:302(XenD27071), l) SEQ ID NO:303 (XenD27072), li) SEQ ID NO:304(XenD27073), lii) SEQ ID NO:305 (XenD27074), liii) SEQ ID NO:306(XenD27075), liv) SEQ ID NO:307 (XenD27076), lv) SEQ ID NO:308(XenD27077) lvi) SEQ ID NO:309 (XenD27078), lvii) SEQ ID NO:317(XenD28173), lviii) SEQ ID NO:318 (XenD24876), lix) SEQ ID NO:320(XenD27162), lx) SEQ ID NO:321 (XenD27163), lxi) SEQ ID NO:323(XenD27164), lxii) SEQ ID NO:324 (XenD27165) lxiii) SEQ ID NO:357(XENP31582 Chain 1), lxiv) SEQ ID NO:358 (XENP31583 Chain 1), lxv) SEQID NO:359 (XENP31584 Chain 1), lxvi) SEQ ID NO:380 (XENP32187 Chain 1),lxvii) SEQ ID NO:381 (XENP32188 Chain 1), lxviii) SEQ ID NO:382(XENP32189, Chain 1), lxix) SEQ ID NO:425 (XENP32190 Chain 1), lxx) SEQID NO:384 (XENP32191 Chain 1), lxxi) SEQ ID NO:385 (XENP32991 Chain 1),lxxii) SEQ ID NO:386 (XENP32992 Chain 1), lxxiii) SEQ ID NO:387(XENP32993 Chain 1), lxxiv) SEQ ID NO:388 (XENP32994 Chain 1), lxxv) SEQID NO:389 (XENP32995 Chain 1), lxxvi) SEQ ID NO:390 (XENP32996 Chain 1),lxxvi) SEQ ID NO:391 (XENP32997 Chain 1), lxxvii) SEQ ID NO:392(XENP32998 Chain 1), lxxvii) SEQ ID NO:393 (XENP32999 Chain 1), lxxviii)SEQ ID NO:394 (XENP33000 Chain 1), lxxix) SEQ ID NO:395 (XENP33001 Chain1), lxxx) SEQ ID NO:396 (XENP33002 Chain 1), lxxxi) SEQ ID NO:397(XENP33003 Chain 1), lxxxii) SEQ ID NO:398 (XENP33004 Chain 1), lxxxiii)SEQ ID NO:426 (XENP33005 Chain 1), lxxxiv) SEQ ID NO:427 (XENP33006Chain 1), lxxxv) SEQ ID NO:428 (XENP33007 Chain 1), lxxxvi) SEQ IDNO:429 (XENP33008 Chain 1), lxxxvii) SEQ ID NO:429 (XENP33008 Chain 1),lxxxviii) SEQ ID NO:430 (XENP33009 Chain 1), lxxxix) SEQ ID NO:431(XENP33010 Chain 1), and xc) SEQ ID NO:383 (XENP33011 Chain 1); and saidsecond fusion protein having a polypeptide sequence selected from thegroup consisting of: i) SEQ ID NO:48 (XENP27201 Chain 2), ii) SEQ IDNO:126 (XenD24779), iii) SEQ ID NO:127 (XenD24780), iv) SEQ ID NO:128(XenD24781), v) SEQ ID NO:129 (XenD24782), vi) SEQ ID NO:130(XenD24783), vii) SEQ ID NO:131 (XenD24784), viii) SEQ ID NO:132(XenD24785), ix) SEQ ID NO:133 (XenD24786), x) SEQ ID NO:134(XenD24787), xi) SEQ ID NO:135 (XenD24788), xii) SEQ ID NO:136(XenD24789), xiii) SEQ ID NO:137 (XenD24790), xiv) SEQ ID NO:138(XenD24791), xv) SEQ ID NO:236 (XenD25911), xvi) SEQ ID NO:237(XenD25912), xvii) SEQ ID NO:238 (XenD25913), xviii) SEQ ID NO:239(XenD25914), xix) SEQ ID NO:240 (XenD25915), xx) SEQ ID NO:241(XenD25916), xxi) SEQ ID NO:242 (XenD25917), xxii) SEQ ID NO:243(XenD25918), xxiii) SEQ ID NO:244 (XenD25919), xxiv) SEQ ID NO:245(XenD25920), xxv) SEQ ID NO:246 (XenD25921), xxvi) SEQ ID NO:299(XenD26427), xxvii) SEQ ID NO:300 (XenD26428), xxviii) SEQ ID NO:311(XenD27089), xxix) SEQ ID NO:312 (XenD27090), xxx) SEQ ID NO:313(XenD27091), xxxi) SEQ ID NO:314 (XenD27092), xxxii) SEQ ID NO:315(XenD27093), xxxiii) SEQ ID NO:316 (XenD27094), xxxix) SEQ ID NO:319(XenD24877), xl) SEQ ID NO:322 (XenD27166), xli) SEQ ID NO:421(XENP31582 Chain 2), xlii) SEQ ID NO:422 (XENP31583 Chain 2), xliii) SEQID NO:423 (XENP31584 Chain 2), xliv) SEQ ID NO:402 (XENP32187 Chain 2),xlv) SEQ ID NO:403 (XENP32188 Chain 2), xlvi) SEQ ID NO:404 (XENP32189,Chain 2), xlvii) SEQ ID NO:405 (XENP32190 Chain 2), xlviii) SEQ IDNO:406 (XENP32191 Chain 2), xlix) SEQ ID NO:407 (XENP32991 Chain 2), 1)SEQ ID NO:408 (XENP32992 Chain 2), li) SEQ ID NO:409 (XENP32993 Chain2), lii) SEQ ID NO:410 (XENP32994 Chain 2), liii) SEQ ID NO:411(XENP32995 Chain 2), liv) SEQ ID NO:412 (XENP32996 Chain 2), lv) SEQ IDNO:413 (XENP32997 Chain 2), lvi) SEQ ID NO:414 (XENP32998 Chain 2),lvii) SEQ ID NO:415 (XENP32999 Chain 2), lviii) SEQ ID NO:416 (XENP33000Chain 2), lix) SEQ ID NO:417 (XENP33001 Chain 2), lx) SEQ ID NO:418(XENP33002 Chain 2), lxi) SEQ ID NO:419 (XENP33003 Chain 2), lxii) SEQID NO:420 (XENP33004 Chain 2), lxiii) SEQ ID NO:399 (XENP33005 Chain 2),lxiv) SEQ ID NO:400 (XENP33006 Chain 2), lxv) SEQ ID NO:401 (XENP33007Chain 2), lxvi) SEQ ID NO:289 (XENP33008 Chain 2), lxvii) SEQ ID NO:289(XENP33008 Chain 2), lxviii) SEQ ID NO:290 (XENP33009 Chain 2), lxix)SEQ ID NO:352 (XENP33010 Chain 2), and lxx) SEQ ID NO:354 (XENP33011Chain 2).

Preferred embodiments include XENP31251, XENP31254, XENP31258,XENP32186, XENP32187, XENP32188, XENP32189, XENP32190, and XENP32191. Infurther embodiments, XENP31251, XENP31254, XENP31258, XENP32186,XENP32187, XENP32188, XENP32189, XENP32190, and XENP32191 includefurther glycoengineering.

IX. Nucleic Acids of the Invention

The invention further provides nucleic acid compositions encoding theheterodimeric Fc fusion protein, the IL-12 subunits, and the IL-12heterodimeric complex of the invention (or, in the case of a monomer Fcdomain protein, nucleic acids encoding those as well).

As will be appreciated by those in the art, the nucleic acidcompositions will depend on the format of the heterodimeric protein.Thus, for example, when the format requires three amino acid sequences,three nucleic acid sequences can be incorporated into one or moreexpression vectors for expression. Similarly for some formats, only twonucleic acids are needed; again, they can be put into one or twoexpression vectors.

As is known in the art, the nucleic acids encoding the components of theinvention can be incorporated into expression vectors as is known in theart, and depending on the host cells used to produce the heterodimericFc fusion proteins of the invention. Generally the nucleic acids areoperably linked to any number of regulatory elements (promoters, originof replication, selectable markers, ribosomal binding sites, inducers,etc.). The expression vectors can be extra-chromosomal or integratingvectors.

The nucleic acids and/or expression vectors of the invention are thentransformed into any number of different types of host cells as is wellknown in the art, including mammalian, bacterial, yeast, insect and/orfungal cells, with mammalian cells (e.g. CHO cells), finding use in manyembodiments.

In some embodiments, nucleic acids encoding each monomer, as applicabledepending on the format, are each contained within a single expressionvector, generally under different or the same promoter controls. Inembodiments of particular use in the present invention, each of thesetwo or three nucleic acids are contained on a different expressionvector.

The heterodimeric Fc fusion protein of the invention are made byculturing host cells comprising the expression vector(s) as is wellknown in the art. Once produced, traditional fusion protein or antibodypurification steps are done, including an ion exchange chromotographystep. As discussed herein, having the pIs of the two monomers differ byat least 0.5 can allow separation by ion exchange chromatography orisoelectric focusing, or other methods sensitive to isoelectric point.That is, the inclusion of pI substitutions that alter the isoelectricpoint (pI) of each monomer so that each monomer has a different pI andthe heterodimer also has a distinct pI, thus facilitating isoelectricpurification of the heterodimer (e.g., anionic exchange chromatography,cationic exchange chromatography). These substitutions also aid in thedetermination and monitoring of any contaminating homodimerspost-purification (e.g., IEF gels, cIEF, and analytical IEX columns).

X. Biological and Biochemical Functionality of IL-12 HeterodimericImmunomodulatory Fc Fusion Proteins

Generally the Fc fusion proteins of the invention are administered topatients with cancer, and efficacy is assessed, in a number of ways asdescribed herein. Thus, while standard assays of efficacy can be run,such as cancer load, size of tumor, evaluation of presence or extent ofmetastasis, etc., immuno-oncology treatments can be assessed on thebasis of immune status evaluations as well. This can be done in a numberof ways, including both in vitro and in vivo assays. For example,evaluation of changes in immune status (e.g., presence of ICOS+ CD4+ Tcells following ipi treatment) along with “old fashioned” measurementssuch as tumor burden, size, invasiveness, LN involvement, metastasis,etc. can be done. Thus, any or all of the following can be evaluated:the inhibitory effects of PVRIG on CD4⁺ T cell activation orproliferation, CD8⁺ T (CTL) cell activation or proliferation, CD8⁺ Tcell-mediated cytotoxic activity and/or CTL mediated cell depletion, NKcell activity and NK mediated cell depletion, the potentiating effectsof PVRIG on Treg cell differentiation and proliferation and Treg- ormyeloid derived suppressor cell (MDSC)-mediated immunosuppression orimmune tolerance, and/or the effects of PVRIG on proinflammatorycytokine production by immune cells, e.g., IL-2, IFN-γ or TNF-αproduction by T or other immune cells.

In some embodiments, assessment of treatment is done by evaluatingimmune cell proliferation, using for example, CFSE dilution method, Ki67intracellular staining of immune effector cells, and ³H-thymidineincorporation method,

In some embodiments, assessment of treatment is done by evaluating theincrease in gene expression or increased protein levels ofactivation-associated markers, including one or more of: CD25, CD69,CD137, ICOS, PD1, GITR, OX40, and cell degranulation measured by surfaceexpression of CD107A.

In general, gene expression assays are done as is known in the art.

In general, protein expression measurements are also similarly done asis known in the art.

In some embodiments, assessment of treatment is done by assessingcytotoxic activity measured by target cell viability detection viaestimating numerous cell parameters such as enzyme activity (includingprotease activity), cell membrane permeability, cell adherence, ATPproduction, co-enzyme production, and nucleotide uptake activity.Specific examples of these assays include, but are not limited to,Trypan Blue or PI staining, ⁵¹Cr or ³⁵S release method, LDH activity,MTT and/or WST assays, Calcein-AM assay, Luminescent based assay, andothers.

In some embodiments, assessment of treatment is done by assessing T cellactivity measured by cytokine production, measure either intracellularlyin culture supernatant using cytokines including, but not limited to,IFNγ, TNFα, GM-CSF, IL2, IL6, IL4, IL5, IL10, IL13 using well knowntechniques.

Accordingly, assessment of treatment can be done using assays thatevaluate one or more of the following: (i) increases in immune response,(ii) increases in activation of αβ and/or γδ T cells, (iii) increases incytotoxic T cell activity, (iv) increases in NK and/or NKT cellactivity, (v) alleviation of αβ and/or γδ T-cell suppression, (vi)increases in pro-inflammatory cytokine secretion, (vii) increases inIL-2 secretion; (viii) increases in interferon-γ production, (ix)increases in Th1 response, (x) decreases in Th2 response, (xi) decreasesor eliminates cell number and/or activity of at least one of regulatoryT cells (Tregs).

A. Assays to Measure Efficacy and Potency

In some embodiments, T cell activation is assessed using a MixedLymphocyte Reaction (MLR) assay as is known in the art. An increase inactivity indicates immunostimulatory activity. Appropriate increases inactivity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in immune response as measured for an example byphosphorylation or de-phosphorylation of different factors, or bymeasuring other post translational modifications. IL-12 mediates IFNγexpression and secretion through phosphorylation of STAT4 (Morinobu etal., 2002). Accordingly, in a preferred embodiment, the signalingpathway assay measures increases or decreases in immune response asindicated by phosphorylation of STAT4. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in activation of αβ and/or γδ T cells as measured for anexample by cytokine secretion or by proliferation or by changes inexpression of activation markers like for an example CD137, CD107a, PD1,etc. An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in cytotoxic T cell activity as measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in NK and/or NKT cell activity as measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by changes in expression of activation markerslike for an example CD107a, etc. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in αβ and/or γδ T-cell suppression, as measured for an exampleby cytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in pro-inflammatory cytokine secretion as measured for exampleby ELISA or by Luminex or by Multiplex bead based methods or byintracellular staining and FACS analysis or by Alispot etc. An increasein activity indicates immunostimulatory activity. Appropriate increasesin activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in IL-2 secretion as measured for example by ELISA or byLuminex or by Multiplex bead based methods or by intracellular stainingand FACS analysis or by Alispot etc. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in interferon-γ production as measured for example by ELISA orby Luminex or by Multiplex bead based methods or by intracellularstaining and FACS analysis or by Alispot etc. An increase in activityindicates immunostimulatory activity. Appropriate increases in activityare outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in Th1 response as measured for an example by cytokinesecretion or by changes in expression of activation markers. An increasein activity indicates immunostimulatory activity. Appropriate increasesin activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in Th2 response as measured for an example by cytokinesecretion or by changes in expression of activation markers. An increasein activity indicates immunostimulatory activity. Appropriate increasesin activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases cell number and/or activity of at least one of regulatory Tcells (Tregs), as measured for example by flow cytometry or by IHC. Adecrease in response indicates immunostimulatory activity. Appropriatedecreases are the same as for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in M2 macrophages cell numbers, as measured for example byflow cytometry or by IHC. A decrease in response indicatesimmunostimulatory activity. Appropriate decreases are the same as forincreases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in M2 macrophage pro-tumorigenic activity, as measured for anexample by cytokine secretion or by changes in expression of activationmarkers. A decrease in response indicates immunostimulatory activity.Appropriate decreases are the same as for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in N2 neutrophils increase, as measured for example by flowcytometry or by IHC. A decrease in response indicates immunostimulatoryactivity. Appropriate decreases are the same as for increases, outlinedbelow.

In one embodiment, the signaling pathway assay measures increases ordecreases in N2 neutrophils pro-tumorigenic activity, as measured for anexample by cytokine secretion or by changes in expression of activationmarkers. A decrease in response indicates immunostimulatory activity.Appropriate decreases are the same as for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in inhibition of T cell activation, as measured for an exampleby cytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in inhibition of CTL activation as measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in αβ and/or γδ T cell exhaustion as measured for an exampleby changes in expression of activation markers. A decrease in responseindicates immunostimulatory activity. Appropriate decreases are the sameas for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases αβ and/or γδ T cell response as measured for an example bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in stimulation of antigen-specific memory responses asmeasured for an example by cytokine secretion or by proliferation or bychanges in expression of activation markers like for an example CD45RA,CCR7 etc. An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in apoptosis or lysis of cancer cells as measured for anexample by cytotoxicity assays such as for an example MTT, Cr release,Calcine AM, or by flow cytometry based assays like for an example CFSEdilution or propidium iodide staining etc. An increase in activityindicates immunostimulatory activity. Appropriate increases in activityare outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in stimulation of cytotoxic or cytostatic effect on cancercells, as measured for an example by cytotoxicity assays such as for anexample MTT, Cr release, Calcine AM, or by flow cytometry based assayslike for an example CFSE dilution or propidium iodide staining etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases direct killing of cancer cells as measured for an example bycytotoxicity assays such as for an example MTT, Cr release, Calcine AM,or by flow cytometry based assays like for an example CFSE dilution orpropidium iodide staining etc. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases Th17 activity as measured for an example by cytokine secretionor by proliferation or by changes in expression of activation markers.An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in induction of complement dependent cytotoxicity and/orantibody dependent cell-mediated cytotoxicity, as measured for anexample by cytotoxicity assays such as for an example MTT, Cr release,Calcine AM, or by flow cytometry based assays like for an example CFSEdilution or propidium iodide staining etc. An increase in activityindicates immunostimulatory activity. Appropriate increases in activityare outlined below.

In one embodiment, T cell activation is measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. ForT-cells, increases in proliferation, cell surface markers of activation(e.g., CD25, CD69, CD137, PD1), cytotoxicity (ability to kill targetcells), and cytokine production (e.g., IL-2, IL-4, IL-6, IFNγ, TNF-α,IL-10, IL-17A) would be indicative of immune modulation that would beconsistent with enhanced killing of cancer cells.

In one embodiment, NK cell activation is measured for example by directkilling of target cells like for an example cancer cells or by cytokinesecretion or by changes in expression of activation markers like for anexample CD107a, etc. For NK cells, increases in proliferation,cytotoxicity (ability to kill target cells and increases CD107a,granzyme, and perforin expression), cytokine production (e.g., IFNγ andTNF), and cell surface receptor expression (e.g. CD25) would beindicative of immune modulation that would be consistent with enhancedkilling of cancer cells.

In one embodiment, γδ T cell activation is measured for example bycytokine secretion or by proliferation or by changes in expression ofactivation markers.

In one embodiment, Th1 cell activation is measured for example bycytokine secretion or by changes in expression of activation markers.

Appropriate increases in activity or response (or decreases, asappropriate as outlined above), are increases of 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95% or 98 to 99% percent over the signal ineither a reference sample or in control samples, for example testsamples that do not contain an IL-12 heterodimeric fusion protein of theinvention. Similarly, increases of at least one-, two-, three-, four- orfive-fold as compared to reference or control samples show efficacy.

XI. Combination Therapy

In some aspects, the IL-12-Fc fusion proteins described herein isadministered in combination with another therapeutic agent. Administered“in combination”, as used herein, means that two (or more) differenttreatments are delivered to the subject during the course of thesubject's affliction with the disorder, e.g., the two or more treatmentsare delivered after the subject has been diagnosed with the disorder andbefore the disorder has been cured or eliminated or treatment has ceasedfor other reasons. In some embodiments, the delivery of one treatment isstill occurring when the delivery of the second begins, so that there isoverlap in terms of administration. This is sometimes referred to hereinas “simultaneous” or “concurrent delivery”. In other embodiments, thedelivery of one treatment ends before the delivery of the othertreatment begins. In some embodiments of either case, the treatment ismore effective because of combined administration. For example, thesecond treatment is more effective, e.g., an equivalent effect is seenwith less of the second treatment, or the second treatment reducessymptoms to a greater extent, than would be seen if the second treatmentwere administered in the absence of the first treatment, or theanalogous situation is seen with the first treatment. In someembodiments, delivery is such that the reduction in a symptom, or otherparameter related to the disorder is greater than what would be observedwith one treatment delivered in the absence of the other. The effect ofthe two treatments can be partially additive, wholly additive, orgreater than additive. The delivery can be such that an effect of thefirst treatment delivered is still detectable when the second isdelivered.

The IL-12-Fc fusion proteins (e.g., any XENP sequence described herein)described herein and the at least one additional therapeutic agent canbe administered simultaneously, in the same or in separate compositions,or sequentially. For sequential administration, the IL-12-Fc fusionproteins (e.g., any XENP sequence described herein) described herein canbe administered first, and the additional agent can be administeredsecond, or the order of administration can be reversed.

The IL-12-Fc fusion proteins (e.g., any XENP sequence described herein)described herein and/or other therapeutic agents, procedures ormodalities can be administered during periods of active disorder, orduring a period of remission or less active disease. The IL-12-Fc fusionproteins (e.g., any XENP sequence described herein) can be administeredbefore the other treatment, concurrently with the treatment,post-treatment, or during remission of the disorder.

A. Checkpoint Blockade Antibodies

In some embodiments, the IL-12-Fc fusion proteins described herein arecombined with other therapeutic agents including checkpoint blockadeantibodies, such as but not limited to, a PD-1 inhibitor, a TIM3inhibitor, a CTLA4 inhibitor, a PD-L1 inhibitor, a TIGIT inhibitor, aLAG3 inhibitor, or a combination thereof.

1. Anti-PD-1 Antibodies

In some embodiments, an IL-12-Fc fusion protein described herein can beadministered to a subject with cancer in combination with an anti-PD-1antibody. In some cases, the anti-PD-1 antibody includes XENP16432 (abivalent anti-PD-1 mAb, a checkpoint inhibitor which enhances anti-tumoractivity by de-repressing the engrafted human T cells; sequencesdepicted in FIG. 53 ).

In some embodiments, the Il-12-Fc fusion proteins of the invention areadministered in combination with Keytruda® or Optivo®.

Exemplary non-limiting anti-PD-1 antibody molecules are disclosed in US2015/0210769, published on Jul. 30, 2015, entitled “Antibody Moleculesto PD-1 and Uses Thereof,” incorporated by reference in its entirety.

In one embodiment, the anti-PD-1 antibody molecule includes at least oneor two heavy chain variable domain (optionally including a constantregion), at least one or two light chain variable domain (optionallyincluding a constant region), or both, comprising the amino acidsequence of BAP049-Clone-A, BAP049-Clone-B, BAP049-Clone-C,BAP049-Clone-D, or BAP049-Clone-E; or as described in Table 1 of US2015/0210769, or encoded by the nucleotide sequence in Table 1; or asequence substantially identical (e.g., at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or higher identical) to any of the aforesaidsequences. The anti-PD-1 antibody molecule, optionally, comprises aleader sequence from a heavy chain, a light chain, or both, as shown inTable 4 of US 2015/0210769; or a sequence substantially identicalthereto.

In yet another embodiment, the anti-PD-1 antibody molecule includes atleast one, two, or three complementarity determining regions (CDRs) froma heavy chain variable region and/or a light chain variable region of anantibody described herein, e.g., an antibody chosen from any ofBAP049-hum01, BAP049-hum02, BAP049-hum03, BAP049-hum04, BAP049-hum05,BAP049-hum06, BAP049-hum07, BAP049-hum08, BAP049-hum09, BAP049-hum10,BAP049-hum11, BAP049-hum12, BAP049-hum13, BAP049-hum14, BAP049-hum15,BAP049-hum16, BAP049-Clone-A, BAP049-Clone-B, BAP049-Clone-C,BAP049-Clone-D, or BAP049-Clone-E; or as described in Table 1, orencoded by the nucleotide sequence in Table 1; or a sequencesubstantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or higher identical) to any of the aforesaid sequences.

In yet another embodiment, the anti-PD-1 antibody molecule includes atleast one, two, or three CDRs (or collectively all of the CDRs) from aheavy chain variable region comprising an amino acid sequence shown inTable 1 of US 2015/0210769, or encoded by a nucleotide sequence shown inTable 1. In one embodiment, one or more of the CDRs (or collectively allof the CDRs) have one, two, three, four, five, six or more changes,e.g., amino acid substitutions or deletions, relative to the amino acidsequence shown in Table 1, or encoded by a nucleotide sequence shown inTable 1.

In yet another embodiment, the anti-PD-1 antibody molecule includes atleast one, two, or three CDRs (or collectively all of the CDRs) from alight chain variable region comprising an amino acid sequence shown inTable 1 of US 2015/0210769, or encoded by a nucleotide sequence shown inTable 1. In one embodiment, one or more of the CDRs (or collectively allof the CDRs) have one, two, three, four, five, six or more changes,e.g., amino acid substitutions or deletions, relative to the amino acidsequence shown in Table 1, or encoded by a nucleotide sequence shown inTable 1. In certain embodiments, the anti-PD-1 antibody moleculeincludes a substitution in a light chain CDR, e.g., one or moresubstitutions in a CDR1, CDR2 and/or CDR3 of the light chain. In oneembodiment, the anti-PD-1 antibody molecule includes a substitution inthe light chain CDR3 at position 102 of the light variable region, e.g.,a substitution of a cysteine to tyrosine, or a cysteine to serineresidue, at position 102 of the light variable region according to Table1 (e.g., SEQ ID NO: 16 or 24 for murine or chimeric, unmodified; or anyof SEQ ID NOs: 34, 42, 46, 54, 58, 62, 66, 70, 74, or 78 for a modifiedsequence).

In another embodiment, the anti-PD-1 antibody molecule includes at leastone, two, three, four, five or six CDRs (or collectively all of theCDRs) from a heavy and light chain variable region comprising an aminoacid sequence shown in Table 1 of US 2015/0210769, or encoded by anucleotide sequence shown in Table 1. In one embodiment, one or more ofthe CDRs (or collectively all of the CDRs) have one, two, three, four,five, six or more changes, e.g., amino acid substitutions or deletions,relative to the amino acid sequence shown in Table 1, or encoded by anucleotide sequence shown in Table 1.

In one embodiment, the anti-PD-1 antibody molecule includes:

(a) a heavy chain variable region (VH) comprising a VHCDR1 amino acidsequence of SEQ ID NO: 4, a VHCDR2 amino acid sequence of SEQ ID NO: 5,and a VHCDR3 amino acid sequence of SEQ ID NO: 3; and a light chainvariable region (VL) comprising a VLCDR1 amino acid sequence of SEQ IDNO: 13, a VLCDR2 amino acid sequence of SEQ ID NO: 14, and a VLCDR3amino acid sequence of SEQ ID NO: 33, each disclosed in Table 1 of US2015/0210769;

(b) a VH comprising a VHCDR1 amino acid sequence chosen from SEQ ID NO:1; a VHCDR2 amino acid sequence of SEQ ID NO: 2; and a VHCDR3 amino acidsequence of SEQ ID NO: 3; and a VL comprising a VLCDR1 amino acidsequence of SEQ ID NO: 10, a VLCDR2 amino acid sequence of SEQ ID NO:11, and a VLCDR3 amino acid sequence of SEQ ID NO: 32, each disclosed inTable 1 of US 2015/0210769;

(c) a VH comprising a VHCDR1 amino acid sequence of SEQ ID NO: 224, aVHCDR2 amino acid sequence of SEQ ID NO: 5, and a VHCDR3 amino acidsequence of SEQ ID NO: 3; and a VL comprising a VLCDR1 amino acidsequence of SEQ ID NO: 13, a VLCDR2 amino acid sequence of SEQ ID NO:14, and a VLCDR3 amino acid sequence of SEQ ID NO: 33, each disclosed inTable 1 of US 2015/0210769; or

(d) a VH comprising a VHCDR1 amino acid sequence of SEQ ID NO: 224; aVHCDR2 amino acid sequence of SEQ ID NO: 2; and a VHCDR3 amino acidsequence of SEQ ID NO: 3; and a VL comprising a VLCDR1 amino acidsequence of SEQ ID NO: 10, a VLCDR2 amino acid sequence of SEQ ID NO:11, and a VLCDR3 amino acid sequence of SEQ ID NO: 32, each disclosed inTable 1 of US 2015/0210769.

In another embodiment, the anti-PD-1 antibody molecule comprises (i) aheavy chain variable region (VH) comprising a VHCDR1 amino acid sequencechosen from SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 224; a VHCDR2amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5; and a VHCDR3 aminoacid sequence of SEQ ID NO: 3; and (ii) a light chain variable region(VL) comprising a VLCDR1 amino acid sequence of SEQ ID NO: 10 or SEQ IDNO: 13, a VLCDR2 amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 14,and a VLCDR3 amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33, eachdisclosed in Table 1 of US 2015/0210769.

In other embodiments, the PD-1 inhibitor is an anti-PD-1 antibody chosenfrom nivolumab, pembrolizumab, or pidilizumab.

In some embodiments, the anti-PD-1 antibody is nivolumab. Alternativenames for nivolumab include MDX-1106, MDX-1106-04, ONO-4538, orBMS-936558. In some embodiments, the anti-PD-1 antibody is Nivolumab(CAS Registry Number: 946414-94-4). Nivolumab is a fully human IgG4monoclonal antibody which specifically blocks PD1. Nivolumab (clone 5C4)and other human monoclonal antibodies that specifically bind to PD1 aredisclosed in U.S. Pat. No. 8,008,449 and WO2006/121168. In oneembodiment, the inhibitor of PD-1 is nivolumab, and having a sequencedisclosed herein (or a sequence substantially identical or similarthereto, e.g., a sequence at least 85%, 90%, 95% identical or higher tothe sequence specified). In some embodiments, the anti-PD-1 antibody ispembrolizumab. Pembrolizumab (also referred to as lambrolizumab,MK-3475, MK03475, SCH-900475 or KEYTRUDA®; Merck) is a humanized IgG4monoclonal antibody that binds to PD-1. Pembrolizumab and otherhumanized anti-PD-1 antibodies are disclosed in Hamid, O. et al. (2013)New England Journal of Medicine 369 (2): 134-44, U.S. Pat. No. 8,354,509and WO2009/114335.

In one embodiment, the inhibitor of PD-1 is pembrolizumab disclosed in,e.g., U.S. Pat. No. 8,354,509 and WO 2009/114335, and having a sequencedisclosed herein (or a sequence substantially identical or similarthereto, e.g., a sequence at least 85%, 90%, 95% identical or higher tothe sequence specified).

In some embodiments, the anti-PD-1 antibody is pidilizumab. Pidilizumab(CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that bindsto PD1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodiesare disclosed in U.S. Pat. No. 8,747,847 and WO2009/101611.

Other anti-PD1 antibodies include AMP 514 (Amplimmune), among others,e.g., anti-PD1 antibodies disclosed in U.S. Pat. No. 8,609,089, US2010028330, and/or US 20120114649.

In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., animmunoadhesin comprising an extracellular or PD-1 binding portion ofPD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of animmunoglobulin sequence). In some embodiments, the PD-1 inhibitor isAMP-224 (B7-DCIg; Amplimmune; e.g., disclosed in WO2010/027827 andWO2011/066342), is a PD-L2 Fc fusion soluble receptor that blocks theinteraction between PD-1 and B7-H1.

In some embodiments, anti-PD-1 antibodies can be used in combinationwith an IL-12 Fc fusion protein of the invention. There are severalanti-PD-1 antibodies including, but not limited to, two currently FDAapproved antibodies, pembrolizumab and nivolizumab, as well as those inclinical testing currently, including, but not limited to, tislelizumab,Sym021, REGN2810 (developed by Rengeneron), JNJ-63723283 (developed by Jand J), SHR-1210, pidilizumab, AMP-224, MEDIo680, PDR001 and CT-001, aswell as others outlined in Liu et al., J. Hemat. & Oncol. (2017)10:136,the antibodies therein expressly incorporated by reference.

In some embodiments, an IL-12-Fc fusion protein described herein can beused in combination with a PD-1 inhibitor (e.g., an anti-PD-1 antibody).In certain embodiments, an IL-12-Fc fusion protein (e.g., any XENPsequence described herein) described herein is administered incombination with an anti-PD-1 antibody.

2. Anti-TIM3 Antibodies

In some embodiments, the IL-12-Fc fusion proteins of the invention areadministered in combination with an anti-TIM3 antibody.

In some embodiments, the Il-12-Fc fusion proteins of the invention areadministered in combination with LY3321367 (Eli Lilly and Company),MBG453 (Novartis Pharmaceuticals), and TSR-022 (Tesaro, Inc.).

Exemplary non-limiting anti-TIM-3 antibody molecules are disclosed in US2015/0218274, published on Aug. 6, 2015, entitled “Antibody Molecules toTIM-3 and Uses Thereof,” incorporated by reference in its entirety.

In one embodiment, the anti-TIM-3 antibody molecule includes at leastone or two heavy chain variable domain (optionally including a constantregion), at least one or two light chain variable domain (optionallyincluding a constant region), or both, comprising the amino acidsequence of ABTIM3, ABTIM3-hum01, ABTIM3-hum02, ABTIM3-hum03,ABTIM3-hum04, ABTIM3-hum05, ABTIM3-hum06, ABTIM3-hum07, ABTIM3-hum08,ABTIM3-hum09, ABTIM3-hum10, ABTIM3-hum11, ABTIM3-hum12, ABTIM3-hum13,ABTIM3-hum14, ABTIM3-hum15, ABTIM3-hum16, ABTIM3-hum17, ABTIM3-hum18,ABTIM3-hum19, ABTIM3-hum20, ABTIM3-hum21, ABTIM3-hum22, ABTIM3-hum23; oras described in Tables 1-4 of US 2015/0218274; or encoded by thenucleotide sequence in Tables 1-4; or a sequence substantially identical(e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higheridentical) to any of the aforesaid sequences. The anti-TIM-3 antibodymolecule, optionally, comprises a leader sequence from a heavy chain, alight chain, or both, as shown in US 2015/0218274; or a sequencesubstantially identical thereto.

In yet another embodiment, the anti-TIM-3 antibody molecule includes atleast one, two, or three complementarity determining regions (CDRs) froma heavy chain variable region and/or a light chain variable region of anantibody described herein, e.g., an antibody chosen from any of ABTIM3,ABTIM3-hum01, ABTIM3-hum02, ABTIM3-hum03, ABTIM3-hum04, ABTIM3-hum05,ABTIM3-hum06, ABTIM3-hum07, ABTIM3-hum08, ABTIM3-hum09, ABTIM3-hum10,ABTIM3-hum11, ABTIM3-hum12, ABTIM3-hum13, ABTIM3-hum14, ABTIM3-hum15,ABTIM3-hum16, ABTIM3-hum17, ABTIM3-hum18, ABTIM3-hum19, ABTIM3-hum20,ABTIM3-hum21, ABTIM3-hum22, ABTIM3-hum23; or as described in Tables 1-4of US 2015/0218274; or encoded by the nucleotide sequence in Tables 1-4;or a sequence substantially identical (e.g., at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaidsequences.

In yet another embodiment, the anti-TIM-3 antibody molecule includes atleast one, two, or three CDRs (or collectively all of the CDRs) from aheavy chain variable region comprising an amino acid sequence shown inTables 1-4 of US 2015/0218274, or encoded by a nucleotide sequence shownin Tables 1-4. In one embodiment, one or more of the CDRs (orcollectively all of the CDRs) have one, two, three, four, five, six ormore changes, e.g., amino acid substitutions or deletions, relative tothe amino acid sequence shown in Tables 1-4, or encoded by a nucleotidesequence shown in Table 1-4.

In yet another embodiment, the anti-TIM-3 antibody molecule includes atleast one, two, or three CDRs (or collectively all of the CDRs) from alight chain variable region comprising an amino acid sequence shown inTables 1-4 of US 2015/0218274, or encoded by a nucleotide sequence shownin Tables 1-4. In one embodiment, one or more of the CDRs (orcollectively all of the CDRs) have one, two, three, four, five, six ormore changes, e.g., amino acid substitutions or deletions, relative tothe amino acid sequence shown in Tables 1-4, or encoded by a nucleotidesequence shown in Tables 1-4. In certain embodiments, the anti-TIM-3antibody molecule includes a substitution in a light chain CDR, e.g.,one or more substitutions in a CDR1, CDR2 and/or CDR3 of the lightchain.

In another embodiment, the anti-TIM-3 antibody molecule includes atleast one, two, three, four, five or six CDRs (or collectively all ofthe CDRs) from a heavy and light chain variable region comprising anamino acid sequence shown in Tables 1-4 of US 2015/0218274, or encodedby a nucleotide sequence shown in Tables 1-4. In one embodiment, one ormore of the CDRs (or collectively all of the CDRs) have one, two, three,four, five, six or more changes, e.g., amino acid substitutions ordeletions, relative to the amino acid sequence shown in Tables 1-4, orencoded by a nucleotide sequence shown in Tables 1-4.

In one embodiment, the anti-TIM-3 antibody molecule includes:

-   -   (a) a heavy chain variable region (VH) comprising a VHCDR1 amino        acid sequence chosen from SEQ ID NO: 9; a VHCDR2 amino acid        sequence of SEQ ID NO: 10; and a VHCDR3 amino acid sequence of        SEQ ID NO: 5; and a light chain variable region (VL) comprising        a VLCDR1 amino acid sequence of SEQ ID NO: 12, a VLCDR2 amino        acid sequence of SEQ ID NO: 13, and a VLCDR3 amino acid sequence        of SEQ ID NO: 14, each disclosed in Tables 1-4 of US        2015/0218274;    -   (b) a VH comprising a VHCDR1 amino acid sequence chosen from SEQ        ID NO: 3; a VHCDR2 amino acid sequence of SEQ ID NO: 4; and a        VHCDR3 amino acid sequence of SEQ ID NO: 5; and a VL comprising        a VLCDR1 amino acid sequence of SEQ ID NO: 6, a VLCDR2 amino        acid sequence of SEQ ID NO: 7, and a VLCDR3 amino acid sequence        of SEQ ID NO: 8, each disclosed in Tables 1-4 of US        2015/0218274;    -   (c) a VH comprising a VHCDR1 amino acid sequence chosen from SEQ        ID NO: 9; a VHCDR2 amino acid sequence of SEQ ID NO: 25; and a        VHCDR3 amino acid sequence of SEQ ID NO: 5; and a VL comprising        a VLCDR1 amino acid sequence of SEQ ID NO: 12, a VLCDR2 amino        acid sequence of SEQ ID NO: 13, and a VLCDR3 amino acid sequence        of SEQ ID NO: 14, each disclosed in Tables 1-4 of US        2015/0218274;    -   (d) a VH comprising a VHCDR1 amino acid sequence chosen from SEQ        ID NO: 3; a VHCDR2 amino acid sequence of SEQ ID NO: 24; and a        VHCDR3 amino acid sequence of SEQ ID NO: 5; and a VL comprising        a VLCDR1 amino acid sequence of SEQ ID NO: 6, a VLCDR2 amino        acid sequence of SEQ ID NO: 7, and a VLCDR3 amino acid sequence        of SEQ ID NO: 8, each disclosed in Tables 1-4 of US        2015/0218274;    -   (e) a VH comprising a VHCDR1 amino acid sequence chosen from SEQ        ID NO: 9; a VHCDR2 amino acid sequence of SEQ ID NO: 31; and a        VHCDR3 amino acid sequence of SEQ ID NO: 5; and a VL comprising        a VLCDR1 amino acid sequence of SEQ ID NO: 12, a VLCDR2 amino        acid sequence of SEQ ID NO: 13, and a VLCDR3 amino acid sequence        of SEQ ID NO: 14, each disclosed in Tables 1-4 of US        2015/0218274; or    -   (f) a VH comprising a VHCDR1 amino acid sequence chosen from SEQ        ID NO: 3; a VHCDR2 amino acid sequence of SEQ ID NO: 30; and a        VHCDR3 amino acid sequence of SEQ ID NO: 5; and a VL comprising        a VLCDR1 amino acid sequence of SEQ ID NO: 6, a VLCDR2 amino        acid sequence of SEQ ID NO: 7, and a VLCDR3 amino acid sequence        of SEQ ID NO: 8, each disclosed in Tables 1-4 of US        2015/0218274.

Exemplary anti-TIM-3 antibodies are disclosed in U.S. Pat. No.8,552,156, WO 2011/155607, EP 2581113 and U.S. Publication No.:2014/044728.

In some embodiments, anti-TIM-3 antibodies can be used in combination anIL-12 Fc fusion protein of the invention. There are several TIM-3antibodies in clinical development, including, but not limited to,MBG453 and TSR-022.

In some embodiments, an IL-12-Fc fusion protein described herein can beused in combination with a TIM-3 inhibitor (e.g., an anti-TIM3antibody). In certain embodiments, an IL-12 Fc fusion protein (e.g., anyXENP sequence described herein) described herein is administered incombination with an anti-TIM3 antibody.

3. Anti-CTLA4 Antibodies

Exemplary anti-CTLA4 antibodies include tremelimumab (IgG2 monoclonalantibody available from Pfizer, formerly known as ticilimumab,CP-675,206); and ipilimumab (CTLA-4 antibody, also known as MDX-010, CASNo. 477202-00-9). Other exemplary anti-CTLA-4 antibodies are disclosed,e.g., in U.S. Pat. No. 5,811,097.

In one embodiment, the anti-CTLA4 antibody is ipilimumab disclosed in,e.g., U.S. Pat. Nos. 5,811,097, 7,605,238, WO00/32231 and WO97/20574,and having a sequence disclosed herein (or a sequence substantiallyidentical or similar thereto, e.g., a sequence at least 85%, 90%, 95%identical or higher to the sequence specified).

In one embodiment, the anti-CTLA4 antibody is tremelimumab disclosed in,e.g., U.S. Pat. No. 6,682,736 and WO00/37504, and having a sequencedisclosed herein (or a sequence substantially identical or similarthereto, e.g., a sequence at least 85%, 90%, 95% identical or higher tothe sequence specified).

In some embodiments, anti-CTLA-4 antibodies can be used in combinationwith an IL-12-Fc fusion protein of the invention. Thus, suitableanti-CTLA-4 antibodies for use in combination therapies as outlinedherein include, but are not limited to, one currently FDA approvedantibody ipilimumab, and several more in development, includingCP-675,206 and AGEN-1884.

In some embodiments, an IL-12-Fc fusion protein described herein can beused in combination with a CTLA-4 inhibitor (e.g., an anti-CTLA-4antibody). In certain embodiments, an IL-12-Fc fusion proteins (e.g.,any XENP sequence described herein) described herein is administered incombination with an anti-CTLA-4 antibody.

4. Anti-PD-L1 Antibodies

Exemplary non-limiting anti-PD-L1 antibody molecules are disclosed in US2016/0108123, published on Apr. 21, 2016, entitled “Antibody Moleculesto PD-L1 and Uses Thereof,” incorporated by reference in its entirety.

In one embodiment, the anti-PD-L1 antibody molecule includes at leastone or two heavy chain variable domain (optionally including a constantregion), at least one or two light chain variable domain (optionallyincluding a constant region), or both, comprising the amino acidsequence of any of BAP058-hum01, BAP058-hum02, BAP058-hum03,BAP058-hum04, BAP058-hum05, BAP058-hum06, BAP058-hum07, BAP058-hum08,BAP058-hum09, BAP058-hum10, BAP058-hum11, BAP058-hum12, BAP058-hum13,BAP058-hum14, BAP058-hum15, BAP058-hum16, BAP058-hum17, BAP058-Clone-K,BAP058-Clone-L, BAP058-Clone-M, BAP058-Clone-N, or BAP058-Clone-O; or asdescribed in Table 1 of US 2016/0108123, or encoded by the nucleotidesequence in Table 1; or a sequence substantially identical (e.g., atleast 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to anyof the aforesaid sequences.

In yet another embodiment, the anti-PD-L1 antibody molecule includes atleast one, two, or three complementarity determining regions (CDRs) froma heavy chain variable region and/or a light chain variable region of anantibody described herein, e.g., an antibody chosen from any ofBAP058-hum01, BAP058-hum02, BAP058-hum03, BAP058-hum04, BAP058-hum05,BAP058-hum06, BAP058-hum07, BAP058-hum08, BAP058-hum09, BAP058-hum10,BAP058-hum11, BAP058-hum12, BAP058-hum13, BAP058-hum14, BAP058-hum15,BAP058-hum16, BAP058-hum17, BAP058-Clone-K, BAP058-Clone-L,BAP058-Clone-M, BAP058-Clone-N, or BAP058-Clone-O; or as described inTable 1 of US 2016/0108123, or encoded by the nucleotide sequence inTable 1; or a sequence substantially identical (e.g., at least 80%, 85%,90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of theaforesaid sequences.

In yet another embodiment, the anti-PD-L1 antibody molecule includes atleast one, two, or three CDRs (or collectively all of the CDRs) from aheavy chain variable region comprising an amino acid sequence shown inTable 1 of US 2016/0108123, or encoded by a nucleotide sequence shown inTable 1. In one embodiment, one or more of the CDRs (or collectively allof the CDRs) have one, two, three, four, five, six or more changes,e.g., amino acid substitutions or deletions, relative to the amino acidsequence shown in Table 1, or encoded by a nucleotide sequence shown inTable 1.

In yet another embodiment, the anti-PD-L1 antibody molecule includes atleast one, two, or three CDRs (or collectively all of the CDRs) from alight chain variable region comprising an amino acid sequence shown inTable 1 of US 2016/0108123, or encoded by a nucleotide sequence shown inTable 1. In one embodiment, one or more of the CDRs (or collectively allof the CDRs) have one, two, three, four, five, six or more changes,e.g., amino acid substitutions or deletions, relative to the amino acidsequence shown in Table 1, or encoded by a nucleotide sequence shown inTable 1. In certain embodiments, the anti-PD-L1 antibody moleculeincludes a substitution in a light chain CDR, e.g., one or moresubstitutions in a CDR1, CDR2 and/or CDR3 of the light chain.

In another embodiment, the anti-PD-L1 antibody molecule includes atleast one, two, three, four, five or six CDRs (or collectively all ofthe CDRs) from a heavy and light chain variable region comprising anamino acid sequence shown in Table 1, or encoded by a nucleotidesequence shown in Table 1 of US 2016/0108123. In one embodiment, one ormore of the CDRs (or collectively all of the CDRs) have one, two, three,four, five, six or more changes, e.g., amino acid substitutions ordeletions, relative to the amino acid sequence shown in Table 1, orencoded by a nucleotide sequence shown in Table 1.

In one embodiment, the anti-PD-L1 antibody molecule includes:

(i) a heavy chain variable region (VH) including a VHCDR1 amino acidsequence chosen from SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 195; aVHCDR2 amino acid sequence of SEQ ID NO: 2; and a VHCDR3 amino acidsequence of SEQ ID NO: 3, each disclosed in Table 1 of US 2016/0108123;and

(ii) a light chain variable region (VL) including a VLCDR1 amino acidsequence of SEQ ID NO: 9, a VLCDR2 amino acid sequence of SEQ ID NO: 10,and a VLCDR3 amino acid sequence of SEQ ID NO: 11, each disclosed inTable 1 of US 2016/0108123.

In another embodiment, the anti-PD-L1 antibody molecule includes:

(i) a heavy chain variable region (VH) including a VHCDR1 amino acidsequence chosen from SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 195; aVHCDR2 amino acid sequence of SEQ ID NO: 5, and a VHCDR3 amino acidsequence of SEQ ID NO: 3, each disclosed in Table 1 of US 2016/0108123;and

(ii) a light chain variable region (VL) including a VLCDR1 amino acidsequence of SEQ ID NO: 12, a VLCDR2 amino acid sequence of SEQ ID NO:13, and a VLCDR3 amino acid sequence of SEQ ID NO: 14, each disclosed inTable 1 of US 2016/0108123.

In one embodiment, the anti-PD-L1 antibody molecule comprises the VHCDR1amino acid sequence of SEQ ID NO: 1. In another embodiment, theanti-PD-L1 antibody molecule comprises the VHCDR1 amino acid sequence ofSEQ ID NO: 4. In yet another embodiment, the anti-PD-L1 antibodymolecule comprises the VHCDR1 amino acid sequence of SEQ ID NO: 195,each disclosed in Table 1 of US 2016/0108123.

In some embodiments, the PD-L1 inhibitor is an antibody molecule. Insome embodiments, the anti-PD-L1 inhibitor is chosen from YW243.55.S70,MPDL3280A, MEDI-4736, MSB-0010718C, MDX-1105, atezolizumab, durbalumab,avelumab, or BMS936559.

In some embodiments, the anti-PD-L1 antibody is atezolizumab.Atezolizumab (also referred to as MPDL3280A and Atezo®; Roche) is amonoclonal antibody that binds to PD-L1. Atezolizumab and otherhumanized anti-PD-L1 antibodies are disclosed in U.S. Pat. No.8,217,149, and having a sequence disclosed herein (or a sequencesubstantially identical or similar thereto, e.g., a sequence at least85%, 90%, 95% identical or higher to the sequence specified).

In some embodiments, the anti-PD-L1 antibody is avelumab. Avelumab (alsoreferred to as A09-246-2; Merck Serono) is a monoclonal antibody thatbinds to PD-L1. Avelumab and other humanized anti-PD-L1 antibodies aredisclosed in U.S. Pat. No. 9,324,298 and WO2013/079174, and having asequence disclosed herein (or a sequence substantially identical orsimilar thereto, e.g., a sequence at least 85%, 90%, 95% identical orhigher to the sequence specified).

In some embodiments, the anti-PD-L1 antibody is durvalumab. Durvalumab(also referred to as MEDI4736; AstraZeneca) is a monoclonal antibodythat binds to PD-L1. Durvalumab and other humanized anti-PD-L1antibodies are disclosed in U.S. Pat. No. 8,779,108, and having asequence disclosed herein (or a sequence substantially identical orsimilar thereto, e.g., a sequence at least 85%, 90%, 95% identical orhigher to the sequence specified).

In some embodiments, the anti-PD-L1 antibody is BMS-936559. BMS-936559(also referred to as MDX-1105; BMS) is a monoclonal antibody that bindsto PD-L1. BMS-936559 and other humanized anti-PD-L1 antibodies aredisclosed in U.S. Pat. No. 7,943,743 and WO2007005874, and having asequence disclosed herein (or a sequence substantially identical orsimilar thereto, e.g., a sequence at least 85%, 90%, 95% identical orhigher to the sequence specified).

In some embodiments, anti-PD-L1 antibodies can be used in combinationwith an IL-12-Fc fusion protein of the invention. There are severalanti-PD-L1 antibodies including three currently FDA approved antibodies,atezolizumab, avelumab, durvalumab, as well as those in clinical testingcurrently, including, but not limited to, LY33000054 and CS1001, as wellas others outlined in Liu et al., J. Hemat. & Oncol. (2017)10:136, theantibodies therein expressly incorporated by reference.

In some embodiments, an IL-12-Fc fusion protein described herein can beused in combination with a PD-L1 or PD-L2 inhibitor (e.g., an anti-PD-L1antibody).

5. Anti-TIGIT Antibodies

In some embodiments, the anti-TIGIT antibody is OMP-313M32. OMP-313M32(OncoMed Pharmaceuticals) is a monoclonal antibody that binds to TIGIT.OMP-313M32 and other humanized anti-TIGIT antibodies are disclosed inUS20160376365 and WO2016191643, and having a sequence disclosed herein(or a sequence substantially identical or similar thereto, e.g., asequence at least 85%, 90%, 95% identical or higher to the sequencespecified).

In some embodiments, the anti-TIGIT antibody is BMS-986207. BMS-986207(also referred to as ONO-4686; Bristol-Myers Squibb) is a monoclonalantibody that binds to TIGIT. BMS-986207 and other humanized anti-TIGITantibodies are disclosed in US20160176963 and WO2016106302, and having asequence disclosed herein (or a sequence substantially identical orsimilar thereto, e.g., a sequence at least 85%, 90%, 95% identical orhigher to the sequence specified).

In some embodiments, the anti-TIGIT antibody is MTIG7192. MTIG7192(Genentech) is a monoclonal antibody that binds to TIGIT. MTIG7192 andother humanized anti-TIGIT antibodies are disclosed in US2017088613,WO2017053748, and WO2016011264, and having a sequence disclosed herein(or a sequence substantially identical or similar thereto, e.g., asequence at least 85%, 90%, 95% identical or higher to the sequencespecified).

In some embodiments, anti-TIGIT antibodies can be used in combinationwith an IL-12-Fc fusion protein of the invention. There are severalTIGIT antibodies in clinical development, BMS-986207, OMP-313M32 andMTIG7192A.

In some embodiments, an IL-12-Fc fusion protein described herein can beused in combination with a TIGIT inhibitor (e.g., an anti-TIGITantibody). In certain embodiments, an IL-12-Fc fusion protein (e.g., anyXENP sequence described herein) described herein is administered incombination with an anti-TIGIT antibody.

6. Anti-LAG3 Antibodies

Exemplary non-limiting anti-LAG-3 antibody molecules are disclosed in US2015/0259420 published on Sep. 17, 2015, entitled “Antibody Molecules toLAG-3 and Uses Thereof,” incorporated by reference in its entirety.

In one embodiment, the anti-LAG-3 antibody molecule includes at leastone or two heavy chain variable domain (optionally including a constantregion), at least one or two light chain variable domain (optionallyincluding a constant region), or both, comprising the amino acidsequence of any of BAP050-hum01, BAP050-hum02, BAP050-hum03,BAP050-hum04, BAP050-hum05, BAP050-hum06, BAP050-hum07, BAP050-hum08,BAP050-hum09, BAP050-hum10, BAP050-hum11, BAP050-hum12, BAP050-hum13,BAP050-hum14, BAP050-hum15, BAP050-hum16, BAP050-hum17, BAP050-hum18,BAP050-hum19, BAP050-hum20, huBAP050(Ser) (e.g., BAP050-hum0l-Ser,BAP050-hum02-Ser, BAP050-hum03-Ser, BAP050-hum04-Ser, BAP050-hum05-Ser,BAP050-hum06-Ser, BAP050-hum07-Ser, BAP050-hum08-Ser, BAP050-hum09-Ser,BAP050-hum10-Ser, BAP050-hum11-Ser, BAP050-hum12-Ser, BAP050-hum13-Ser,BAP050-hum14-Ser, BAP050-hum15-Ser, BAP050-hum18-Ser, BAP050-hum19-Ser,or BAP050-hum20-Ser), BAP050-Clone-F, BAP050-Clone-G, BAP050-Clone-H,BAP050-Clone-I, or BAP050-Clone-J; or as described in Table 1 of US2015/0259420, or encoded by the nucleotide sequence in Table 1; or asequence substantially identical (e.g., at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or higher identical) to any of the aforesaidsequences.

In yet another embodiment, the anti-LAG-3 antibody molecule includes atleast one, two, or three complementarity determining regions (CDRs) froma heavy chain variable region and/or a light chain variable region of anantibody described herein, e.g., an antibody chosen from any ofBAP050-hum01, BAP050-hum02, BAP050-hum03, BAP050-hum04, BAP050-hum05,BAP050-hum06, BAP050-hum07, BAP050-hum08, BAP050-hum09, BAP050-hum10,BAP050-hum11, BAP050-hum12, BAP050-hum13, BAP050-hum14, BAP050-hum15,BAP050-hum16, BAP050-hum17, BAP050-hum18, BAP050-hum19, BAP050-hum20,huBAP050(Ser) (e.g., BAP050-hum0l-Ser, BAP050-hum02-Ser,BAP050-hum03-Ser, BAP050-hum04-Ser, BAP050-hum05-Ser, BAP050-hum06-Ser,BAP050-hum07-Ser, BAP050-hum08-Ser, BAP050-hum09-Ser, BAP050-hum10-Ser,BAP050-hum11-Ser, BAP050-hum12-Ser, BAP050-hum13-Ser, BAP050-hum14-Ser,BAP050-hum15-Ser, BAP050-hum18-Ser, BAP050-hum19-Ser, orBAP050-hum20-Ser), BAP050-Clone-F, BAP050-Clone-G, BAP050-Clone-H,BAP050-Clone-I, or BAP050-Clone-J; or as described in Table 1 of US2015/0259420, or encoded by the nucleotide sequence in Table 1; or asequence substantially identical (e.g., at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or higher identical) to any of the aforesaidsequences.

In yet another embodiment, the anti-LAG-3 antibody molecule includes atleast one, two, or three CDRs (or collectively all of the CDRs) from aheavy chain variable region comprising an amino acid sequence shown inTable 1 of US 2015/0259420, or encoded by a nucleotide sequence shown inTable 1. In one embodiment, one or more of the CDRs (or collectively allof the CDRs) have one, two, three, four, five, six or more changes,e.g., amino acid substitutions or deletions, relative to the amino acidsequence shown in Table 1, or encoded by a nucleotide sequence shown inTable 1.

In yet another embodiment, the anti-LAG-3 antibody molecule includes atleast one, two, or three CDRs (or collectively all of the CDRs) from alight chain variable region comprising an amino acid sequence shown inTable 1 of US 2015/0259420, or encoded by a nucleotide sequence shown inTable 1. In one embodiment, one or more of the CDRs (or collectively allof the CDRs) have one, two, three, four, five, six or more changes,e.g., amino acid substitutions or deletions, relative to the amino acidsequence shown in Table 1, or encoded by a nucleotide sequence shown inTable 1. In certain embodiments, the anti-PD-L1 antibody moleculeincludes a substitution in a light chain CDR, e.g., one or moresubstitutions in a CDR1, CDR2 and/or CDR3 of the light chain.

In another embodiment, the anti-LAG-3 antibody molecule includes atleast one, two, three, four, five or six CDRs (or collectively all ofthe CDRs) from a heavy and light chain variable region comprising anamino acid sequence shown in Table 1, or encoded by a nucleotidesequence shown in Table 1 of US 2015/0259420. In one embodiment, one ormore of the CDRs (or collectively all of the CDRs) have one, two, three,four, five, six or more changes, e.g., amino acid substitutions ordeletions, relative to the amino acid sequence shown in Table 1, orencoded by a nucleotide sequence shown in Table 1.

In one embodiment, the anti-LAG-3 antibody molecule includes:

(i) a heavy chain variable region (VH) including a VHCDR1 amino acidsequence chosen from SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 286; aVHCDR2 amino acid sequence of SEQ ID NO: 2; and a VHCDR3 amino acidsequence of SEQ ID NO: 3, each disclosed in Table 1 of US 2015/0259420;and

(ii) a light chain variable region (VL) including a VLCDR1 amino acidsequence of SEQ ID NO: 10, a VLCDR2 amino acid sequence of SEQ ID NO:11, and a VLCDR3 amino acid sequence of SEQ ID NO: 12, each disclosed inTable 1 of US 2015/0259420.

In another embodiment, the anti-LAG-3 antibody molecule includes:

(i) a heavy chain variable region (VH) including a VHCDR1 amino acidsequence chosen from SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 286; aVHCDR2 amino acid sequence of SEQ ID NO: 5, and a VHCDR3 amino acidsequence of SEQ ID NO: 3, each disclosed in Table 1 of US 2015/0259420;and

(ii) a light chain variable region (VL) including a VLCDR1 amino acidsequence of SEQ ID NO: 13, a VLCDR2 amino acid sequence of SEQ ID NO:14, and a VLCDR3 amino acid sequence of SEQ ID NO: 15, each disclosed inTable 1 of US 2015/0259420.

In one embodiment, the anti-LAG-3 antibody molecule comprises the VHCDR1amino acid sequence of SEQ ID NO: 1. In another embodiment, theanti-LAG-3 antibody molecule comprises the VHCDR1 amino acid sequence ofSEQ ID NO: 4. In yet another embodiment, the anti-LAG-3 antibodymolecule comprises the VHCDR1 amino acid sequence of SEQ ID NO: 286,each disclosed in Table 1 of US 2015/0259420.

In some embodiments, the anti-LAG-3 antibody is BMS-986016. BMS-986016(also referred to as BMS986016; Bristol-Myers Squibb) is a monoclonalantibody that binds to LAG-3. BMS-986016 and other humanized anti-LAG-3antibodies are disclosed in US 2011/0150892, WO2010/019570, andWO2014/008218.

In some embodiments, the anti-LAG3 antibody is LAG525. LAG525 (alsoreferred to as IMP701; Novartis) is a monoclonal antibody that binds toLAG3. LAG525 and other humanized anti-LAG3 antibodies are disclosed inU.S. Pat. No. 9,244,059 and WO2008132601, and having a sequencedisclosed herein (or a sequence substantially identical or similarthereto, e.g., a sequence at least 85%, 90%, 95% identical or higher tothe sequence specified).

Other exemplary anti-LAG3 antibodies are disclosed, e.g., inUS2011150892 and US2018066054.

In some embodiments, anti-LAG-3 antibodies can be used in combinationwith an IL-12-Fc fusion protein of the invention. There are severalanti-LAG-3 antibodies in clinical development including REGN3767, byRegeneron, BMS-986016 (Bristol-Myers Squibb), MK-4280 (Merck), LAG525(Novartis), and TSR-033 (Tesaro).

In some embodiments, an IL-12-Fc fusion protein described herein can beused in combination with a LAG3 inhibitor (e.g., an anti-LAG3 antibody).In certain embodiments, an IL-12-Fc fusion protein (e.g., any XENPsequence described herein) described herein is administered incombination with an anti-LAG3 antibody.

XII. Treatments

Once made, the compositions of the invention find use in a number ofoncology applications, by treating cancer, generally by promoting T cellactivation (e.g., T cells are no longer suppressed) with the binding ofthe heterodimeric Fc fusion proteins of the invention.

Accordingly, the heterodimeric compositions of the invention find use inthe treatment of these cancers.

A. Fusion Protein Compositions for In Vivo Administration

Formulations of the fusion proteins used in accordance with the presentinvention are prepared for storage by mixing a fusion protein having thedesired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (as generally outlined inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]),in the form of lyophilized formulations or aqueous solutions.

B. Administrative Modalities

The fusion proteins and chemotherapeutic agents of the invention areadministered to a subject, in accord with known methods, such asintravenous administration as a bolus or by continuous infusion over aperiod of time.

B. Treatment Modalities

In the methods of the invention, therapy is used to provide a positivetherapeutic response with respect to a disease or condition. By“positive therapeutic response” is intended an improvement in thedisease or condition, and/or an improvement in the symptoms associatedwith the disease or condition. For example, a positive therapeuticresponse would refer to one or more of the following improvements in thedisease: (1) a reduction in the number of neoplastic cells; (2) anincrease in neoplastic cell death; (3) inhibition of neoplastic cellsurvival; (5) inhibition (i.e., slowing to some extent, preferablyhalting) of tumor growth; (6) an increased patient survival rate; and(7) some relief from one or more symptoms associated with the disease orcondition.

Positive therapeutic responses in any given disease or condition can bedetermined by standardized response criteria specific to that disease orcondition. Tumor response can be assessed for changes in tumormorphology (i.e., overall tumor burden, tumor size, and the like) usingscreening techniques such as magnetic resonance imaging (MRI) scan,x-radiographic imaging, computed tomographic (CT) scan, bone scanimaging, endoscopy, and tumor biopsy sampling including bone marrowaspiration (BMA) and counting of tumor cells in the circulation.

In addition to these positive therapeutic responses, the subjectundergoing therapy may experience the beneficial effect of animprovement in the symptoms associated with the disease.

Treatment according to the present invention includes a “therapeuticallyeffective amount” of the medicaments used. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve a desired therapeutic result.

A therapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the medicaments to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the protein or protein portion areoutweighed by the therapeutically beneficial effects.

A “therapeutically effective amount” for tumor therapy may also bemeasured by its ability to stabilize the progression of disease. Theability of a compound to inhibit cancer may be evaluated in an animalmodel system predictive of efficacy in human tumors.

Alternatively, this property of a composition may be evaluated byexamining the ability of the compound to inhibit cell growth or toinduce apoptosis by in vitro assays known to the skilled practitioner. Atherapeutically effective amount of a therapeutic compound may decreasetumor size, or otherwise ameliorate symptoms in a subject. One ofordinary skill in the art would be able to determine such amounts basedon such factors as the subject's size, the severity of the subject'ssymptoms, and the particular composition or route of administrationselected.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. Parenteral compositions may beformulated in dosage unit form for ease of administration and uniformityof dosage. Dosage unit form as used herein refers to physically discreteunits suited as unitary dosages for the subjects to be treated; eachunit contains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

The specification for the dosage unit forms of the present invention aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

The efficient dosages and the dosage regimens for the heterodimericproteins used in the present invention depend on the disease orcondition to be treated and may be determined by the persons skilled inthe art.

An exemplary, non-limiting range for a therapeutically effective amountof an heterodimeric proteins used in the present invention is about0.1-100 mg/kg.

All cited references are herein expressly incorporated by reference intheir entirety.

Whereas particular embodiments of the invention have been describedabove for purposes of illustration, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

EXAMPLES

Examples are provided below to illustrate the present invention. Theseexamples are not meant to constrain the present invention to anyparticular application or theory of operation. For all constant regionpositions discussed in the present invention, numbering is according tothe EU index as in Kabat (Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, 5th Ed., United States Public Health Service,National Institutes of Health, Bethesda, entirely incorporated byreference). Those skilled in the art of antibodies will appreciate thatthis convention consists of nonsequential numbering in specific regionsof an immunoglobulin sequence, enabling a normalized reference toconserved positions in immunoglobulin families. Accordingly, thepositions of any given immunoglobulin as defined by the EU index willnot necessarily correspond to its sequential sequence.

General and specific scientific techniques are outlined in USPublications 2015/0307629, 2014/0288275 and WO2014/145806, all of whichare expressly incorporated by reference in their entirety andparticularly for the techniques outlined therein.

Example 1: IL-12-Fc Fusion Proteins

As with other cytokines, IL-12 has a short half-life, and high dosetreatment to overcome the short half-life results in systemic toxicity.Additionally, it has also been reported that anti-tumor effect requiressustained induction of IFNγ production by IL-12 (Gollob, J A et al.,2000). Further, the IL-12p40 subunit, as either a monomer or ahomodimer, has been reported to antagonize IL-12 activity by competingfor binding to IL-12 receptors (Gillessen, S et al., 1995); accordingly,it is advantageous to pre-complex the IL-12p40 and IL-12p35 subunits. Inorder to address these two caveats, we engineered the IL-12 heterodimeras Fc fusion proteins (collectively referred to hereon as IL-12-Fcfusions) both to enhance circulation through FcRn-mediated recycling andto pre-complex the IL-12p40 and IL-12p35 subunits.

1A: Engineering IL-12-Fc Fusions in Various Formats

We generated the N-terminal IL-12 heterodimeric Fc fusion or“IL-12-heteroFc” format which comprises the IL-12p40 subunitrecombinantly fused to the N-terminus of one side of a heterodimeric Fcand the IL-12p35 subunit recombinantly fused to N-terminus of the otherside of the heterodimeric Fc (FIG. 8A). The IL-12p40 and IL-12p35subunits may be linked to their respective Fc chains by a domain linker.An illustrative protein of this format is XENP27201, sequences for whichare depicted in FIG. 9 .

We also generated the C-terminal IL-12 heterodimeric Fc fusion or“heteroFc-IL-12” format which comprises the IL-12p40 subunitrecombinantly fused to the C-terminus of one side of a heterodimeric Fcand the IL-12p35 subunit recombinantly fused to the C-terminus of theother side of the heterodimeric Fc (FIG. 8B). The IL-12p30 and IL-12p35subunits may be linked to their respective Fc chains by a domain linker.An illustrative protein of this format is XENP27202, sequences for whichare depicted in FIG. 10 .

We further generated the N-terminal single-chain IL-12-Fc fusion or“scIL-12-Fc” format which comprises a single-chain IL-12 complex (or“scIL-12 complex”) recombinantly fused to the N-terminus of one side ofa heterodimeric Fc (optionally via a domain linker), with the other sideof the molecule being a “Fc-only” or “empty-Fc” heterodimeric Fc (FIG.8C-D). The scIL-12 complex can comprise either IL-12p35 N-terminallylinked to IL-12p40 or IL-12p40 N-terminally linked to IL-12p35,optionally with a domain linker. The order of the two subunits in thescIL-12 complex are designated herein as follows: “scIL-12(p40/p35)”,wherein the IL-12p40 subunit is N-terminally linked to the IL-12p35subunit, or “scIL-12(p35/p40)”, wherein the IL-12p35 is N-terminallylinked to the IL-12p35 subunit. Illustrative proteins of the scIL-12-Fcformat include XENP27203 and XENP27204, sequences for which are depictedin FIG. 11 .

Cartoon schematics for additional IL-12-Fc formats contemplated for useare depicted in FIG. 8 .

1B: Production and Physical Characterization of Illustrative IL-12-FcFusions

1B(a): IL-12-heteroFc

Plasmids coding for the IL-12p35 and IL-12p40 subunits were constructedby standard gene synthesis, followed by subcloning into a pTT5expression vector containing Fc fusion partners (e.g., constant regionsas depicted in FIG. 7 ). Proteins were produced by transienttransfection in HEK293E cells and were purified by a two-steppurification process comprising protein A chromatography (purificationpart 1) followed by anion exchange chromatography (purification part 2).Chromatogram depicting purification part 2 for illustrativeIL-12-heteroFc XENP27201 is depicted in FIG. 12A. The chromatogram showsthe isolation of two peaks (peak A and peak B), which were furthercharacterized by analytical size-exclusion chromatography withmulti-angle light scattering (aSEC-MALS) and analytical anion-exchangechromatography (analytical AIEX) for identity, purity and homogeneity asgenerally described below.

Peaks A and B isolated from purification part 2 for XENP27201 wereanalyzed using aSEC-MALS to deduce their component protein species. Theanalysis was performed on an Agilent 1200 high-performance liquidchromatography (HPLC) system. Samples were injected onto a Superdex™ 20010/300 GL column (GE Healthcare Life Sciences) at 1.0 mL/min using1×PBS, pH 7.4 as the mobile phase at 4° C. for 25 minutes with UVdetection wavelength at 280 nM. MALS was performed on a miniDAWN® TREOS®with an Optilab® T-rEX Refractive Index Detector (Wyatt Technology,Santa Barbara, Cali.). Analysis was performed using Agilent OpenLabChromatography Data System (CDS) ChemStation Edition AIC version C.01.07and ASTRA version 6.1.7.15. Chromatograms depicting aSEC separationprofiles for peaks A and B are depicted in FIG. 12B along with MW ofcomponent species as determined by MALS. The profiles show that peak Acomprises species with molecular weights of ˜299 kD and ˜140 kD, whilepeak B primarily comprises a species with molecular weight of ˜118 kD,which is consistent with the calculated molecular weight of XENP27201(based on amino acid sequence) of 110.4 kDa taking into accountadditional mass contributed by glycans.

The peaks from purification part 2 were also analyzed using analyticalAIEX to further assess the purity and homogeneity of peak B. Theanalysis was performed on an Agilent 1200 high-performance liquidchromatography (HPLC) system. Samples were injected onto a ProteomixSAX-NP5 5 μM non-porous column (Sepax Technologies, Inc., Newark, Del.)at 1.0 mL/min using 0-40% NaCl gradient in 20 mM Tris, pH 8.5 bufferwith UV detection wavelength at 280 nM. Analysis was performed usingAgilent OpenLAB CDS ChemStation Edition AIC version C.01.07.Chromatograms depicting analytical AIEX separation of peaks A and B aredepicted in FIG. 12C. Consistent with the aSEC separation profile forpeak B, the analytical AIEX separation profile for peak B illustratesthe purity and homogeneity of species in peak B. From here on, XENP27201refers to peak B as isolated from purification part 2 as depicted inFIG. 12A.

1B(b): scIL-12(p40/p35)-Fc

Plasmids coding for IL-12p40 recombinantly fused to IL-12p35 via alinker were constructed by standard gene synthesis, followed bysubcloning into a pTT5 expression vector containing Fc fusion partners(e.g., constant regions as depicted in FIG. 7 ). Proteins were producedby transient transfection in HEK293E cells and were purified by atwo-step purification process comprising protein A chromatography(purification part 1) and anion exchange chromatography (purificationpart 2).

Chromatogram depicting purification part 2 for illustrativescIL-12(p40/p35)-Fc fusion XENP27203 is depicted in FIG. 13A. As above,the chromatogram shows the isolation of two peaks, which were furthercharacterized by aSEC-MALS and analytical AIEX for identity, purity andhomogeneity as described in Example 1B(a), chromatograms for which aredepicted in FIGS. 13B-C.

The aSEC separation profiles for peaks A and B isolated frompurification part 2 of XENP27203 show that peak A comprises species withmolecular weights of ˜396 kD, ˜188 kD, and ˜118 kD, while peak Bprimarily comprises a species with molecular weight of ˜118 kD, which isconsistent with the calculated molecular weight of XENP27203 (based onamino acid sequence) of 111.3 kDa taking into account additional masscontributed by glycans. The peaks were also analyzed using analyticalAIEX as described in Example 1B(a) to further investigate the purity andhomogeneity of peak B. Consistent with the aSEC separation profile forpeak B, the analytical AIEX separation profile for peak B (FIG. 13C)illustrates the purity and homogeneity of species in peak B. From hereon, XENP27203 refers to peak B as isolated from anion exchange asdepicted in FIG. 13A.

1C: In Vitro Activity of IL-12-Fc Fusions in Induction of STAT4Phosphorylation

Following binding of cytokines to their receptors, Janus kinases (JAKs)associated with the cytokine receptors phosphorylate STAT proteins whichthen translocate into the nucleus to regulate further downstreamprocesses. In particular, IL-12 mediates IFNγ expression and secretionthrough phosphorylation of STAT4 (Morinobu et al., 2002). Accordingly,the ability of the above described IL-12-heteroFc (XENP27201) andscIL-12(p40/p35)-Fc (XENP27203) to induce STAT4 phosphorylation invarious lymphocyte populations was investigated. Bivalent IL-12p35-Fcfusions and IL-12p40-Fc fusions (cartoon schematics and sequences forwhich are depicted in FIGS. 14-15 ) as well as recombinant IL-12 wereused as controls.

Fresh PBMCs were activated by incubation with plate bound anti-CD3 (100ng/ml) for 3 days. Following activation, PBMCs were then incubated withthe indicated test articles at the indicated concentrations for 15minutes at 37° C. Following incubation, PBMCs were first stained withanti-CD3-BUV395 (UCHT1), anti-CD4-BV605 (RPA-T4), anti-CD8-AF700 (SK1),anti-CD14-APC/Fire750 (M5E2), anti-CD20-PerCP/5.5 (2H7), anti-CD25-BV421(M-A251), and anti-CD56-PE antibodies. Following the first stain, cellswere permeabilized using PerFix EXPOSE (Beckman Coulter, Indianapolis,Ind.). Following permeabilization, cells were stained withanti-anti-CD45RA-BV510 (H1I100), anti-FoxP3-AF488 (259D), andanti-pSTAT4-AF647 (38/p-Stat4) antibodies. Following the secondstaining, the cells were analyzed by flow cytometry to investigate STAT4phosphorylation on various lymphocyte populations. Data depicting pSTAT4MFI on various lymphocyte populations, indicating signaling by theIL-12-Fc fusions via IL-12 receptors, are depicted in FIG. 16 .

The data show that both XENP27201 and XENP27203 were active in inducingSTAT4 phosphorylation in various lymphocyte populations to a similarlevel as that induced by recombinant IL-12, while the bivalentIL-12p40-Fc (XENP27560) and bivalent IL-12p35-Fc (XENP27561) fusionswere inactive. Notably, the two IL-12-Fc fusion formats demonstratedsimilar potency. Additionally, the ability of the purified IL-12-Fcfusion proteins to induce STAT4 phosphorylation in comparison toXENP27560 and XENP27561 confirms that that peak B isolated frompurification part 2 for both XENP27201 and XENP27203 (as described inExample 1B) consisted of the active species comprising the completeIL-12 heterodimer.

Example 2: IL-12 Variants Engineered for Reduced Potency

In order to further prolong half-life as well as reduce potential fortoxicity, we engineered IL-12 variants with decreased binding affinityfor IL-12 receptors as we reasoned that this would decrease the antigensink as well as reduce potency.

2A(a): Engineering IL-12p40 Variants

We first identified W15, P17, D18, A19, P20, G21, M23, L40, D41, Q42,S43, E45, L47, T54, 155, Q56, K58, E59, F60, G61, D62, Y66, and K84 aspotential sites on IL-12p40 for introducing affinity-modulatingsubstitutions (residues depicted in FIG. 17 ).

As a second strategy, by using the QuaSAR package in MOE software, weidentified highly exposed aspartic acid, glutamic acid, asparagine, andglutamine residues (according to the water accessible surface areacalculated using a radius of 1.4 Å for the water molecule and apolyhedral representation for each atom) in the IL-12p40 crystalstructure (PDB code 3HMX) reasoning that these residues may contributeto binding between IL-12p40 and the IL-12 receptors. In particular, wefocused our search on residues at which we could incorporate isostericsubstitutions (i.e. Asn and Asp; and Gln and Glu), with the aim tominimize potential for immunogenicity, Accordingly, we identified E3,D7, E12, D14, D18, E22, D29, E32, E33, D41, Q42, E45, Q56, E59, D62,Q65, E73, E86, D93, D97, E100, N103, E110, D129, D142, Q144, E156, D161,N162, E166, E170, Q172, D174, E187, N200, D209, D214, N218, Q220, N226,Q229, E231, E235, Q256, E262, D265, D270, N281, Q289, and E299 whichhave an ASA score of at least 19 (residues and ASA scores depicted inFIG. 18 ).

As described above, the p40 subunit is shared by IL-12 and IL-23. Blochet al. (2018) reported the crystal structure for IL-23 in complex withIL-23R and Nb22E11. As a third strategy, we reasoned that residues onthe IL-12p40 subunit in contact with IL-23R may also be involved withbinding the analogous receptor IL-12R32 in the IL-12 receptor complex.Accordingly, using the crystal structure reported by Bloch et al. (PDBcode 5MZV) and modeling in MOE software, we identified D87, G88, 189,W90, K104, and F106 as residues on IL-12p40 potentially in contact withIL-12R32 (residues and predicted contact type depicted in FIG. 19 ).

Finally, to ensure that we do not disturb the natural interactionbetween the IL-12p40 and IL-12p35 subunits, we identified the followingresidues on IL-12p40 potentially in contact with IL-12p35 based on thecrystal structure reported by Luo et al. (2010): K102, Y114, A176, C177,P178, A179, A180, E181, S183, P185, S204, F206, R208, T242, P243, S245,Y246, F247, S248, D290, R291, Y292, and Y293.

In view of the above we designed a number of IL-12p40 variants, inparticular as described above, at residue at which isostericsubstitutions could be introduced, with the aim to to reduce theaffinity of the IL-12 heterodimer to IL-12 receptors. Substitutions toremove potential N-glycosylation sites in p40 were also designed toexamine the impact of glycosylation on protein heterogeneity. Sequencesfor illustrative variants are depicted in FIG. 20 . Plasmids coding forthe IL-12p40 variants were constructed by standard gene synthesis,followed by subcloning into a pTT5 expression vector containing Fcfusion partners (e.g., constant regions as depicted in FIG. 7 ),corresponding amino acid sequences for which are depicted in FIG. 21 .

2A(b): Engineering IL-12p35 Variants

As with the second strategy described above, we used the QuaSAR packagein MOE software to identify highly exposed aspartic acid, glutamic acid,asparagine, and glutamine residues in the IL-12p35 crystal structure(PDB code 3HMX) reasoning that these residues may contribute to bindingbetween the IL-12 heterodimeric complex and the IL-12 receptors. Again,we focused here, in particular, on residues at which we couldincorporate isosteric substitutions, to minimize the potential forimmunogenicity. We identified Q35, E38, E46, D55, E67, N71, N76, N85,Q135, Q146, N151, E153, E162, and E163 which have an ASA score of atleast 103 (residues and ASA scores depicted in FIG. 22 ).

As above, to ensure that we do not disturb the natural interactionbetween the IL-12p40 and IL-12p354 subunits, we identified the followingresidues on IL-12p35 potentially in contact with IL-12p40 based on thecrystal structure reported by Luo et al. (2010): Q20, S44, E45, E46,H49, K54, T59, V60, E61, C63, L64, P65, E67, L68, S73, C74, R181, 1182,R183, V185, T186, D188, R189, V190, S192, Y193, and A196.

In view of the above we designed a number of IL-12p35 variants withisosteric substitutions with the aim to reduce the affinity of the IL-12heterodimer to IL-12 receptors. Substitutions to remove potentialN-glycosylation sites in p35 were also designed to examine the impact ofglycosylation on protein heterogeneity. Sequences for illustrativevariants are depicted in FIG. 23 . Plasmids coding for the IL-12p35variants were constructed by standard gene synthesis, followed bysubcloning into a pTT5 expression vector containing Fc fusion partners(e.g., constant regions as depicted in FIG. 7 ), corresponding aminoacid sequences for which are depicted in FIG. 24 .

2a(c): Screening IL-12-Fc Fusions Having Reduced Potency

Illustrative variant IL-12-Fc fusion proteins having the above describedvariant IL-12 subunits were designed with the view to generate IL-12-Fcfusions having reduced affinity for the IL-12 receptors in order toreduce potency, sequences for which are depicted in FIG. 25 . Weproduced and purified the IL-12-Fc fusions as generally described inExample 1B, and investigated their activity in a pSTAT4 assay.

Human PBMCs were activated with 100 ng/ml anti-CD3 (OKT3) for 2 days.Activated PBMCs were then incubated with the indicated test articles at37° C. for 15 minutes. Cells were then stained with anti-CD3-BUV395(UCHT1), anti-CD4-BV605 (RPAT4), anti-CD8-AF700 (SK1), anti-CD25-BV510(M-A251), anti-CD45RA-BV421 (H1I100), anti-CD56-PE (N901),anti-FoxP3-AF488 (259D), and anti-pSTAT4-AF647 (38/p-Stat4) using PerFixEXPOSE kit (Beckman Coulter, Indianapolis, Ind.) and analyzed by flowcytometry.

Data showing induction of STAT4 phosphorylation on CD4⁺CD45RA⁺CD25+ andCD8⁺CD45RA⁺CD25⁺ T cells by IL-12-Fc fusion having IL-12p35 or IL-12p40variants are depicted respectively in FIGS. 26 and 27 . FIG. 28 depictsthe EC50 of the various variant IL-12-Fc fusions and the fold decreasein EC50 relative to WT IL-12-Fc XENP27201. The data show that most ofthe IL-12-Fc fusions comprising variant IL-12p40 subunits or IL-12p30subunits exhibit decreased potency in inducing STAT4 phosphorylation.Notably, E59Q and E235Q substitutions in the IL-12p40 subunit, and E153Qand N151D substitutions in the IL-12p35 substitutions were able toindividually provide over 2-fold reduction in potency for the variantIL-12-Fc fusions. Additionally, IL-12p40 double- and triple-mutantscomprising E59Q provided up to 6.5-fold reduction in potency.

Surprisingly, two variant IL-12-Fc fusions comprising IL-12p40(D87N) orIL-12p40(E262Q) exhibited an increase in potency. While we areinterested in decreasing potency with the aim to decrease antigen sinkand thereby increase the half-life of IL-12-Fc fusions, IL-12-Fc fusionshaving increased potency may find use in other contexts.

2B: Engineering IL-12p40 and IL-12p35 Variants (Round 2)

In order to engineer further IL-12p40 and IL-12p35 variants with the aimto reduce their affinity for the IL-12 receptors and reduce the potencyof the biologically active IL-12 complex, we re-applied the strategiesdescribed in Examples 2A with expanded criteria. For example, we used alower ASA score threshold than that defined in Examples 2A foridentifying IL-12p40 and IL-12p35 residues at which to introducesubstitutions. We also expanded our criteria to include additional aminoacid residues, with a particular focus on larger residues such aslysine, at which introduction of substitutions was more likely todisrupt the interaction of IL-12p35, IL-12p40, and/or the IL-12 complexwith the IL-12 receptors.

Similarly, we introduced non-isosteric substitutions, with a particularfocus on larger residues such as lysine, to disrupt the interaction ofIL-12p35, IL-12p40, and/or the IL-12 complex with the IL-12 receptors.Using these approaches, we further identified D34, K99, K163, K258, andK260 in the IL-12p40 subunit; and N21, E79, Q130, N136, E143, K158, andD165 in the IL-12p35 subunit as residues suitable for engineeringsubstitutions. In additional, we generated additional combinationvariants incorporating substitutions identified in Example 2 whichcontributed the greatest reduction in potency.

In view of the above, we designed further IL-12p40 and IL-12p35variants, illustrative sequences for which are depicted respectively inFIGS. 29 and 31 . As above, plasmids coding for the IL-12p40 andIL-12p35 variants were constructed by standard gene synthesis, followedby subcloning into a pTT5 expression vector containing Fc fusionpartners, corresponding amino acid sequences for which are depictedrespectively in FIGS. 30 and 32 . IL-12-Fc fusion proteins weregenerated with the additional variant IL-12 subunits, sequences forwhich are depicted in FIG. 33 , and produced as generally described inExample 1B.

The activity of the additional variant IL-12-Fc fusions wereinvestigated in a pSTAT4 assay as generally described above. Inparticular, human PBMCs were activated with 100 ng/ml anti-CD3 (OKT3)for 2 days. Activated PBMCs were then incubated with the indicated testarticles at 37° C. for 15 minutes. Cells were then stained withanti-CD3-BUV395 (UCHT1), anti-CD4-BV605 (RPAT4), anti-CD8-AF700 (SK1),anti-CD25-BV510 (M-A251), anti-CD45RA-BV421 (H1I100), and anti-CD56-PE(N901). Next, cells were permeabilized using PerFix EXPOSE (BeckmanCoulter, Indianapolis, Ind.). Following permeabilization, cells werestained with anti-FoxP3-AF488 (259D) and anti-pSTAT4-AF647 (38/p-Stat4)and analyzed by flow cytometry. Data depicting pSTAT4 MFI onCD4⁺CD45RA⁺CD25+ and CD8⁺CD45RA⁺CD25⁺ T cells are depicted in FIGS.34-35 , and data depicting the EC50 (and fold decrease relative to WTIL-12-Fc XENP27201) are shown in FIG. 36 . The data show that most ofthe IL-12-Fc fusions comprising variant IL-12p40 and/or IL-12p30subunits exhibit decreased potency in inducing STAT4 phosphorylation.

2C: Engineering IL-12p40 and IL-12p35 Variants (Round 3)

In Example 2B, we found that IL-12-Fc fusion XENP29952 comprisingIL-12p40(E59K) enabled a ˜12 fold reduction, in contrast to IL-12-Fcfusion XENP28825 comprising IL-12p40(E59Q) which had only about 2-foldreduction in potency. Additionally, we noted that several double mutantscomprising E59Q in the IL-12p40 subunit demonstrated >3.5 fold reductionin potency (e.g. XENP29953, XENP29954, and XENP29959). Further, we notedthat while XENP28846 comprising IL-12p35(N151D) enabled increasedpotency, IL-12p35(D55Q/N151D) actually enabled ˜2-fold reduction inpotency, which indicated to us that modification of D55 in the IL-12p35subunit contributes greatly to a reduction in potency. Accordingly, tofurther reduce the potency of IL-12-Fc fusions, we generated additionalcombination variants incorporating substitutions in Example 2B whichcontributed the greatest reduction in potency. In addition as in Example2B, we further included non-isosteric substitutions, with a particularfocus on larger residues such as lysine.

In view of the above, we designed further IL-12p40 and IL-12p35variants, illustrative sequences for which are depicted respectively inFIGS. 37 and 38 . IL-12-Fc fusion proteins were generated with theadditional variant IL-12 subunits, sequences for which are depicted inFIG. 39 , and produced as generally described in Example 1B. Theactivity of the additional variant IL-12-Fc fusions were investigated ina pSTAT4 assay as generally described above. Data depicting pSTAT4 MFIon CD4⁺CD45RA⁺CD25+ and CD8⁺CD45RA⁺CD25⁺ T cells are depicted in FIG. 40, and data depicting the EC50 (and fold decrease relative to WT IL-12-FcXENP27201) are shown in FIG. 41 . The data show that all of the newIL-12-Fc fusions comprising variant IL-12p40 and/or IL-12p30 subunitsexhibited substantially decreased potency in inducing STAT4phosphorylation. Notably, XENP30607 and XENP30608 exhibited ˜100-folddecrease in potency in comparison to XENP27201.

2D: Engineering IL-12p40 and IL-12p35 Variants (Round 4)

We further engineered the IL-12p40 and IL-12p35 subunits both byincorporating additional variants found in Example 2 contribute to ashift in potency, and by further using non-isosteric substitutions, witha particular focus on larger residues such as lysine and tyrosine, todisrupt the interaction of IL-12p35, IL-12p40, and/or the IL-12 complexwith the IL-12 receptors.

In addition, we performed further structural analysis in the MOEsoftware based on surface exposure to identify additional residues inthe IL-12p40 and IL-12p35 subunits at which to introduce modifications.We further identified residues R159 and K264 in the IL-12p40 subunit;and residues L75, L89, F96, M97, L124, M125, and I171 in the IL-12p35subunit. We used the earlier described approaches, as well as alaninesubstitutions (in line with the alanine scanning mutagenesis technique),to preliminarily investigate the effect of modifications at theseadditionally identified residues.

In view of the above, we designed further IL-12p40 and IL-12p35variants, illustrative sequences for which are depicted respectively inFIGS. 42 and 43 . IL-12-Fc fusion proteins were generated with theadditional variant IL-12 subunits, sequences for which are depicted inFIG. 44 , and produced as generally described in Example 1B.

The activity of the additional variant IL-12-Fc fusions wereinvestigated in a pSTAT4 assay as generally described above. Datadepicting pSTAT4 MFI on CD4⁺CD45RA⁺CD25+, CD4⁺CD45RA⁻CD25+,CD8⁺CD45RA⁺CD25+, and CD8⁺CD45RA⁻CD25⁺ T cells are depicted in FIG. 46 ,and data depicting the EC50 (and fold decrease relative to WT IL-12-FcXENP27201) are shown in FIG. 47 . The data show that all of the newIL-12-Fc fusions comprising variant IL-12p40 and/or IL-12p35 subunitsexhibited substantially decreased potency in inducing STAT4phosphorylation.

Example 3: Bivalent IL-12-Fc Fusions

We also conceived bivalent IL-12-Fc fusions. One such format is thebivalent N-terminal IL-12-Fc fusion format (FIGS. 48A-B) which comprisestwo identical monomers each comprising a scIL-12 complex recombinantfused to the N-terminus of a homodimeric Fc chain (optionally via adomain linker). Another such format is the bivalent C-terminal IL-12-Fcfusion format (FIGS. 48C-D) which comprises two identical monomers eachcomprising a scIL-12 complex recombinant fused to the C-terminus of ahomodimeric Fc chain (optionally via a domain linker).

Example 4: Reduced Potency IL-12 Variants in Alternative IL-12-Fc FusionFormats

We also investigated the IL-12 variants in the context of additionalIL-12-Fc fusion formats. Fusions in the scIL-12(p40/p35)-Fc and(scIL-12(p40/p35))₂-Fc formats were generated with either WT IL-12p40subunits or variant IL-12p40(E59K/K99E) subunits and produced asgenerally described in Example 1B (and sequences for which are depictedin FIGS. 49-50 ).

The activity of the IL-12-Fc fusions were investigated in a pSTAT4 assayas generally described above. In particular, human PBMCs were activatedwith 100 ng/ml anti-CD3 (OKT3) for 2 days at 37° C. Activated PBMCs werethen incubated with the indicated test articles for 15 minutes at 37° C.Cells were then stained with anti-CD3-BUV395 (UCHT1), anti-CD4-BV605(RPAT4), anti-CD8-AF700 (SK1), anti-CD25-BV711 (M-A251),anti-CD45RA-BV421 (H1100), and anti-CD56-PE (N901). Next, cells werepermeabilized using PerFix EXPOSE (Beckman Coulter, Indianapolis, Ind.).Following permeabilization, cells were stained with anti-FoxP3-V450(259D) and anti-pSTAT4-AF647 (38/p-Stat4) and analyzed by flowcytometry. Data depicting pSTAT4 MFI on CD4⁺CD45RA⁺CD25+,CD4⁺CD45RA⁻CD25+, CD8⁺CD45RA⁺CD25+, and CD8⁺CD45RA-CD25⁺ T cells aredepicted in FIG. 51 , and data depicting the EC50 (and fold decreaserelative to WT IL-12-Fc XENP27201) are shown in FIG. 52 . The data showthat IL-12-Fc fusions in the various format demonstrated very similarpotency, and the IL-12-Fc fusions in scIL-12(p40/p35)-Fc and(scIL-12(p40/p35))₂-Fc fusions comprising variant IL-12p40(E59K/K99E)subunits demonstrated very similar potency to IL-12-Fc fusions in theIL-12-heteroFc format comprising variant IL-12p40(E59K/K99E) subunit.

Example 5: IL-12-Fc Fusions Enhance Allogeneic Anti-Tumor Effect of TCells In Vivo

Next, we investigated in vivo anti-tumor effect of the IL-12-Fc fusionsof the invention. NOD SCID gamma (NSG) mice were engrafted intradermallywith 3×10⁶ pp-65 expressing MCF-7 cells in the right flank on Day −15.On Day 0, mice were engrafted intraperitoneally with 1.5×10⁶ humanPBMCs. Mice (n=15) were then treated on Days 0, 7, 14, and 21 with 0.03mg/kg XENP29952, a reduced potency IL-12-Fc fusion in the IL-12-heteroFcformat comprising variant IL-12p40(E59K). Controls (n=10) used were PBSand XENP16432 (a bivalent anti-PD-1 mAb, a checkpoint inhibitor whichenhances anti-tumor activity by de-repressing the engrafted human Tcells; sequences depicted in FIG. 53 ). Tumor volumes were monitored bycaliper measurements, data for which are shown (days post 1^(st) dose)in FIG. 54 . Blood and serum were drawn on Days 7, 14, and 21 andanalyzed by flow cytometry to investigate expansion of humanlymphocytes, data for which are depicted in FIG. 55 , as well as byU-PLEX Biomarker Group 1 Human Assays (Meso Scale, Rockville, Md.) forserum cytokine concentrations, data for which are depicted in FIG. 56 .

The data show that the IL-12-heteroFc fusion XENP29952 had significantlyenhanced expansion of CD45⁺, CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells,and NK cells by Day 14 in comparison to both PBS control and checkpointblockade by XENP16432 (statistics performed on log-transformed datausing unpaired t-test). Notably, XENP29952 significantly enhancedanti-tumor activity by Day 11 as indicated by change in tumor volume(statistics performed on baseline corrected data using unpaired t-test).Furthermore, XENP29952 significantly enhanced secretion of IFNγ and CD25by Day 7 in comparison to checkpoint blockade by XENP16432 (statisticsperformed on log-transformed date using unpaired t-test).

In addition to anti-tumor activity, the engrafted human PBMCs develop anautoimmune response against mouse cells and subsequentlygraft-versus-host disease (GVHD). Accordingly, it should be noted thatwhile all the animals treated with XENP29952 were dead by Day 19, thiswas likely due to their succumbing to GVHD exacerbated by significantlyenhanced expansion of human lymphocytes. This highlights the importanceof reduced potency IL-12 variants not just for improvingpharmacokinetics but also for improving therapeutic index.

Example 6: IL-12-Fc Fusions Demonstrate Modulated Activity In VivoCorrelating to In Vitro Potency

As all the animals treated with XENP29952 in Example 5 were dead as aresult of GVHD, we investigated the in vivo activity of IL-12-Fc fusionsengineered with reduced potency IL-12 variants in a GVHD study.

NSG mice were engrafted with 10×10⁶ human PBMCs via IV-OSP on Day −1 anddosed intraperitoneally on Days 0, 7, 14, and 21 with the following testarticles: XENP29952 (a reduced potency IL-12-Fc fusion in theIL-12-heteroFc format comprising variant IL-12p40(E59K)), XENP30597 (areduced potency IL-12-Fc fusion in the IL-12-heteroFc format comprisingvariant IL-12p40(E59K/K99E)), XENP31254 (a reduced potency IL-12-Fcfusion in the IL-12-heteroFc format comprising variantIL-12p40(D18K/E59K/K99E)), XENP31251 (a reduced potency IL-12-Fc fusionin the IL-12-heteroFc format comprising variant IL-12p40(E59K/K99Y)), orXENP31258 (a reduced potency IL-12-Fc fusion in the IL-12-heteroFcformat comprising variant IL-12p40(E59K/K99E/K264E)) at 0.3 or 0.03mg/kg. Body weights were assessed twice per week as an indicator ofGVHD, data for which are depicted in FIG. 57 as a change in body weight(relative to initial body weight). Additionally, blood was drawn on Day7, 10, 14, and 31 to investigate the activation of various T cellpopulations as indicated by PD-1 expression levels (data for which aredepicted in FIGS. 58 and 59 ), and serum was drawn to investigatecytokine secretion (data for which are depicted in FIG. 60 ). Notably,the data show a dose response for the test articles (i.e. enhanced GVHD,T cell activation, and IFNγ secretion by 0.3 mg/kg dose in comparison to0.03 mg/kg dose).

It was surprising that XENP31251, which appeared to be one of theweakest variants in inducing STAT4 phosphorylation on CD4⁺ T cells invitro (see FIGS. 46A-46B), was one of the stronger inducers of GVHD.Accordingly, we re-investigated the in vitro activity of the sameillustrative reduced potency IL-12-Fc in a STAT4 phosphorylation assayas generally described above using two separate PBMC donors. Human PBMCswere activated with 1 μg/ml anti-CD3 (OKT3) for 2 days at 37° C.Activated PBMCs were then incubated with the indicated test articles at37° C. for 15 minutes.

Cells were then stained with anti-CD3-BUV395 (UCHT1), anti-CD4-BV605(RPAT4), anti-CD8-AF700 (SK1), anti-CD25-BV510 (M-A251),anti-CD45-BV785, anti-CD45RA-BV421 (H1I100), anti-CD16-PE (B73), andanti-CD56-PE (N901). Next, cells were permeabilized using PerFix EXPOSE(Beckman Coulter, Indianapolis, Ind.). Following permeabilization, cellswere stained with anti-FoxP3-AF488 (259D) and anti-pSTAT4-AF647(38/p-Stat4) and analyzed by flow cytometry. Induction of STAT4phosphorylation on various lymphocyte populations in PBMCs from the twodonors are depicted in FIG. 61 (data not shown for the second donor butincorporated by reference from FIG. 62 in 62/848,512). The data fromboth donors and across the various lymphocyte populations show a potencyladder with XENP29952 as the most potent variant, XENP31254 andXENP31258 as the least potent variants, and XENP30597 and XENP31251falling in between. Notably, the degree of GVHD, T cell activation, andIFNγ secretion as induced by the reduced potency IL-12-Fc fusionvariants in vivo correlated with the in vitro potency. For example, at0.03 mg/kg dose, XENP29952 induced greater GVHD, T cell activation, andIFNγ secretion than all the reduced potency IL-12-Fc fusions, whileXENP31254 and XENP31258 induced the least GVHD, T cell activation, andIFNγ secretion.

Example 7: IL-12-Fc Fusions have Anti-Tumor Activity and CombineProductively with Checkpoint Blockade

Next, we investigated the in vivo anti-tumor effect of combining theadditional IL-12-Fc fusions as well as the effect of combining theIL-12-Fc fusions with checkpoint blockade. NSG mice were engraftedintradermally with 3×10⁶ pp-65 expressing MCF-7 cells in the right flankon Day −15. On Day 0, mice were engrafted intraperitoneally with 5×10⁶human PBMCs. Mice were then treated on Days 0, 7, 14, and 21 with 0.1mg/kg XENP31251 alone or in combination with 3.0 mg/kg anti-PD-1 mAbXENP16432. Controls used were PBS, 3.0 mg/kg XENP16432 alone, andXENP31258. Tumor volumes were monitored by caliper measurements, datafor which are shown (days post 1^(st) dose) in FIG. 62 . Blood was drawnon Days 7, 14, and 21 and analyzed by flow cytometry to investigateexpansion of human lymphocytes, data for which are depicted in FIG. 63for Day 14.

The data show that XENP31258 significantly enhanced anti-tumor activityby Day 14, and XENP31251 (alone or in combination with XENP16432)significantly enhanced anti-tumor activity (as indicated by change intumor volume) by Day 16 in comparison to treatment with PBS (statisticsperformed on baseline corrected data using Mann-Whitney test). Notably,the data show that XENP31251 in combination with XENP16432 significantlyenhanced anti-tumor activity by Day 21 in comparison to treatment withXENP16432 alone; and that treatment with XENP31251 in combination withXENP16432 significantly enhanced lymphocyte expansion in comparison toeither XENP31251 or XENP16432 alone, indicating that IL-12-Fc fusionscombine productively with checkpoint blockade.

Example 8: Removing Free Cysteine in IL-12p40 Subunit

The IL-12p40 subunit has a free cysteine at position 252 (numberedaccording to the Human IL-12 subunit beta (IL-12p40) mature formsequence as depicted in FIG. 1 ) which may bond with other freecysteines leading at least to heterogeneity and at worse toimmunogenicity. Accordingly, IL-12p40 variants were engineered to removethe free cysteine, for example, by introducing C252S modification(although other substitutions may also be used). Modification of C252(e.g. C252S) can be used alone or in combination with any other IL-12p40variants, such as affinity or expression variants. Illustrative IL-12p40variants comprising a modification at C252 to remove the free cysteineare depicted in FIG. 65 . Illustrative IL-12-Fc fusions proteins weregenerated with the additional variant IL-12p40 subunits, sequences forwhich are depicted in FIG. 66 , and produced as generally described inExample 1B.

The activity of the IL-12p40 variants engineered to remove the freecysteine were investigated in a pSTAT4 assay as generally describedabove in order to ascertain that removal of the free cysteine did notengender any change in activity. Activated PBMCs were incubated with theindicated test articles and pSTAT4 MFI on various populations wereassessed, data for which are depicted in FIG. 67 . The data show thatthe variants comprising the C252S modification demonstrated similaractivity to the counterpart variant which did not comprise the C252Smodification. Notably, for 3 of the 4 variants tested for which weengineered versions with and without the C252S mutation (i.e.D18K/E59K/K99E; D18K/E59K/K99E/K264E; and E59K/K99E), the C252S mutationappeared to slightly increase the potency of the IL-12-Fc fusions.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the compositions, systems and methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention. Modifications of the above-described modesfor carrying out the invention that are obvious to persons of skill inthe art are intended to be within the scope of the following claims. Allpatents and publications mentioned in the specification are indicativeof the levels of skill of those skilled in the art to which theinvention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

All headings and section designations are used for clarity and referencepurposes only and are not to be considered limiting in any way. Forexample, those of skill in the art will appreciate the usefulness ofcombining various aspects from different headings and sections asappropriate according to the spirit and scope of the invention describedherein.

All references cited herein are hereby incorporated by reference hereinin their entireties and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

Many modifications and variations of this application can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments and examplesdescribed herein are offered by way of example only, and the applicationis to be limited only by the terms of the appended claims, along withthe full scope of equivalents to which the claims are entitled.

What is claimed:
 1. A pharmaceutical composition comprising: a) aheterodimeric Fc fusion protein comprising: i) a first fusion proteincomprising an amino acid sequence as set forth in SEQ ID NO: 382; andii) a second fusion protein comprising an amino acid sequence as setforth in SEQ ID NO: 404, wherein said first fusion protein furthercomprises the amino acid substitutions M428L/N434S as compared to SEQ IDNO:382 and said second fusion protein further comprises the amino acidsubstitutions M428L/N434S as compared to SEQ ID NO:404, wherein thenumbering of each amino acid substitution is according to EU numbering;and b) a pharmaceutically acceptable carrier.